ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2015

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1268

Mechanisms and Inhibition ofEF-G-dependent Translocationand Recycling of the BacterialRibosome

ANNELI BORG

ISSN 1651-6214ISBN 978-91-554-9289-2urn:nbn:se:uu:diva-258990

Dissertation presented at Uppsala University to be publicly examined in B22, BMC,Husargatan 3, Uppsala, Friday, 25 September 2015 at 10:00 for the degree of Doctor ofPhilosophy. The examination will be conducted in English. Faculty examiner: ProfessorRuben Gonzalez (Columbia University, Department of Chemistry).

AbstractBorg, A. 2015. Mechanisms and Inhibition of EF-G-dependent Translocation and Recyclingof the Bacterial Ribosome. Digital Comprehensive Summaries of Uppsala Dissertations fromthe Faculty of Science and Technology 1268. 60 pp. Uppsala: Acta Universitatis Upsaliensis.ISBN 978-91-554-9289-2.

The GTPase elongation factor G (EF-G) is an important player in the complex process of proteinsynthesis by bacterial ribosomes. Although extensively studied much remains to be learnedabout this fascinating protein. In the elongation phase, after incorporation of each amino acidinto the growing peptide chain, EF-G translocates the ribosome along the mRNA template. Inthe recycling phase, when the synthesis of a protein has been completed, EF-G, together withribosome recycling factor (RRF), splits the ribosome into its subunits. We developed the first invitro assay for measuring the average time of a complete translocation step at any position alongthe mRNA. Inside the open reading frame, at saturating EF-G concentration and low magnesiumion concentration, translocation rates were fast and compatible with elongation rates observedin vivo. We also determined the complete kinetic mechanism for EF-G- and RRF-dependentsplitting of the post-termination ribosome. We showed that splitting occurs only when RRFbinds before EF-G and that the rate and GTP consumption of the reaction varies greatly withthe factor concentrations.

The antibiotic fusidic acid (FA) inhibits bacterial protein synthesis by binding to EF-G whenthe factor is ribosome bound, during translocation and ribosome recycling. We developedexperimental methods and a theoretical framework for analyzing the effect of tight-bindinginhibitors like FA on protein synthesis. We found that FA targets three different states duringeach elongation cycle and that it binds to EF-G on the post-termination ribosome both in thepresence and absence of RRF. The stalling time of an FA-inhibited ribosome is about hundred-fold longer than the time of an uninhibited elongation cycle and therefore each binding eventhas a large impact on the protein synthesis rate and may induce queuing of ribosomes on themRNA. Although ribosomes in the elongation and the recycling phases are targeted with similarefficiency, we showed that the main effect of FA in vivo is on elongation. Our results may serveas a basis for modelling of EF-G function and FA inhibition inside the living cell and for structuredetermination of mechanistically important intermediate states in translocation and ribosomerecycling.

Keywords: Protein synthesis, Elongation factor G, Translocation, Ribosome recycling, Fusidicacid

Anneli Borg, Department of Cell and Molecular Biology, Structure and Molecular Biology,596, Uppsala University, SE-751 24 Uppsala, Sweden.

© Anneli Borg 2015

ISSN 1651-6214ISBN 978-91-554-9289-2urn:nbn:se:uu:diva-258990 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-258990)

“Life is just a series of rate constants” John R. Lorsch

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Borg, A., Ehrenberg, M. (2015) Determinants of the rate of

mRNA translocation in bacterial protein synthesis. Journal of Molecular Biology, 427(9):1835-1847

II Borg, A., Holm M., Shiroyama I., Hauryliuk V., Pavlov M., Sanyal S., Ehrenberg M. (2014) Fusidic acid targets Elongation factor G in several stages of translocation on the bacterial ribo-some. The journal of Biological Chemistry, 290(6):3440-3454

III Borg A., Pavlov M., Ehrenberg M. (2015) Complete kinetic

mechanism for recycling of the bacterial ribosome (Submitted manuscript)

IV Borg, A., Pavlov M., Ehrenberg M. (2015) Fusidic acid inhibi-

tion of EF-G- and RRF-promoted recycling of the bacterial ri-bosome (Manuscript)

Reprints were made with permission from the respective publishers.

Contents

Introduction ...........................................................................................11 The bacterial ribosome .......................................................................12

Structure .......................................................................................12 Magnesium ions and the ribosome ..................................................13

Bacterial protein synthesis ..................................................................13 Initiation .......................................................................................14 Elongation .....................................................................................14 Termination...................................................................................16 Recycling ......................................................................................16

The present work....................................................................................18 The in vitro translation system ............................................................19 The quench-flow technique .................................................................19 The stopped-flow technique and Rayleigh light scattering .....................20 Mean time calculations .......................................................................22

EF-G and translocation ...........................................................................26 The pre-translocation complex ............................................................26 GTP hydrolysis and Pi release .............................................................27 Structural studies of EF-G in translocation ...........................................28 Biochemical studies of EF-G in translocation .......................................29

EF-G and ribosome recycling ..................................................................34

EF-G and fusidic acid .............................................................................37

In vitro experiments and the living cell ....................................................42

Future outlook........................................................................................45

Sammanfattning på Svenska....................................................................47

Acknowledgements ................................................................................51

References .............................................................................................53

Abbreviations

A adenosine A site aminoacyl-tRNA site ASL anticodon stem loop ATP adenosine 5’-triphosphate C cytidine cryo-EM cryo-electron microscopy DNA deoxyribonucleic acid E site exit site E. coli Escherichia coli EF-G elongation factor G EF-Tu elongation factor Tu FA fusidic acid fMet formylmethionine FRET Förster resonance energy transfer G guanosine GTP guanosine 5’-triphosphate IF initiation factor Met methionine Mg magnesium mRNA messenger RNA P site peptidyl-tRNA site PEP phosphoenol pyruvate Pi inorganic phosphate RF release factor RNA ribonucleic acid RRF ribosome recycling factor rRNA ribosomal RNA SD sequence Shine-Dalgarno sequence T. thermophilus Thermus thermophilus tRNA transfer RNA U uridine

Introduction

A rapidly growing bacterial cell contains tens of thousands of ribosomes (1), that are responsible for producing all the proteins required for the cell to survive and to reproduce. Proteins perform diverse tasks in the cell, ranging from catalysis of chemical reactions (enzymes) and regulation of gene ex-pression (activators and repressors) to transport of ions across the cell mem-brane (ion channels) and sensing of signals from the cell exterior (receptors). Other examples of protein function are as building blocks in cytoskeletal networks that support cell shape and as motor proteins involved in cell mo-tility and intracellular transport systems. Approximately half of the dry weight of a bacterial cell is made up of proteins (2) and a large fraction of the total energy consumption of the cell is devoted to their synthesis. To maintain fast growth it is therefore vital for the bacterial cell that protein synthesis is both efficient and accurate.

Figure 1. The central dogma of molecular biology

The central dogma of molecular biology describes the fundamental processes that convert a gene sequence in DNA into a functional protein (Figure 1). The bacterial chromosome that carries the genetic information is duplicated by DNA polymerase enzymes in a process called replication and is passed on to each daughter cell. Genes in the DNA are used as templates for synthe-sis of single stranded chains of ribonucleotides, called messenger RNAs (mRNAs), by RNA polymerase enzymes in a process called transcription. A ribosome binds at the 5’-end of the mRNA and interconnects amino acids into protein chains according to the nucleotide sequence of the mRNA, in a process called translation. A set of three consecutive nucleotides in the mRNA, a codon, corresponds to one amino acid in the protein being synthe-sized. To decode the mRNA sequence the ribosome takes help from adaptor molecules, called transfer RNAs (tRNAs), that are single stranded RNA molecules, about 75 nucleotides in length, with an overall L-shaped appear-ance. One end of the tRNA, the acceptor stem, carries an amino acid, loaded

11

onto it by an aminoacyl-tRNA synthetase enzyme. The anticodon stem loop (ASL), at the other end of the L-shaped structure, contains a three-nucleotide anticodon which basepairs with the mRNA codon. There are four types of nucleotides in the mRNA, adenosine (A), cytidine (C), guanosine (G) and uridine (U), and since each codon has three nucleotides there are 43 = 64 different codons. There are only twenty amino acids and most of them are encoded by more than one codon. tRNAs that carry the same amino acid, but read different codons, are called isoacceptors. Since an isoacceptor is in of-ten able to read several codons, some mismatches in the basepairing between the codon and the anticodon, that lead to incorporation of the same amino acid, are accepted by the ribosome.

The bacterial ribosome The ribosome is a large macromolecular complex consisting of RNAs and proteins. It coordinates the activities of mRNA, tRNAs and translation fac-tors (helper proteins) in the complex process of linking amino acids together into functional proteins. The structure and composition of ribosomes varies to some extent between the three domains of life (Bacteria, Archaea and Eukarya). In this section I will describe the overall architecture and the im-portant functional sites of bacterial ribosomes of which the most well studied are from Escherichia coli (E. coli).

Structure A bacterial ribosome consists of three ribosomal RNA (rRNA) molecules and about 50 proteins (3) that build up the small (30S) and large (50S) ribosomal subunits. The two subunits join during protein synthesis to form the complete 70S ribosome. The interface between the ribosomal subunits is mainly com-posed of RNA and the ribosomal proteins are primarily found on the outer surface of the ribosome. There are three tRNA binding sites in the ribosome, made up from partial binding sites on each of the subunits, the aminoacyl-tRNA (A) site, the peptidyl-tRNA (P) site and the exit (E) site (4-6).

The 30S subunit is made up from the 16S rRNA, which assists mRNA bind-ing to the ribosome, and 21 proteins. The 16S rRNA contains the anti-Shine Dalgarno (SD) sequence which base-pairs with the SD sequence situated upstream of the start codon in most bacterial mRNAs (7). This positions the start codon in the partial P site of the 30S subunit, for binding of the initiator tRNA, fMet-tRNAfMet, during initiation of protein synthesis. It is in the A site of the 30S subunit that decoding of the genetic information in the mRNA occurs. The tRNA anticodon basepairs with the mRNA codon and the geom-

12

etry of the codon-anticodon helix is monitored by the ribosome to ensure that the correct tRNA has been selected.

The 50S subunit contains two rRNA molecules, 23S and 5S, surrounded by proteins. The 23S rRNA positions the acceptor ends of the tRNAs in the A and P sites of the 50S subunit by base-pairing with their 3’-terminal nucleo-tides, the CCA ends. Domain V of the 23S rRNA contains the peptidyl trans-ferase center that catalyzes the chemical reaction that adds an amino acid to the growing peptide chain.

Magnesium ions and the ribosome The correct folding of the ribosomal RNA is highly dependent on the coor-dination of both monovalent and divalent metal ions. Magnesium ions are the most abundant multivalent metal cations inside the cell. They are small and have a high charge density, which makes them suitable for neutralizing the negative charges of the phosphate groups of the rRNA backbone to ena-ble its dense packing inside the ribosome (8). In the crystal structure of the 70S ribosome from E. coli more than 170 magnesium ions were observed (9). A number of magnesium ions were involved in the bridging of the two ribosomal subunits and one particular magnesium ion was found to be im-portant for proper mRNA binding to the 30S subunit (10). Magnesium ions are not only vital for the structural stability of the ribosome but also for its function. For example, the association of the small and large subunits in vitro, to form a complete 70S ribosome, is favored at high magnesium ion concentration (11). The accuracy of tRNA selection in the ribosomal A site and the rate of peptide bond formation are also strongly affected (12-14). The efficiency of amino acid incorporation increases at higher magnesium ion concentrations but at the cost of reduced accuracy (13). In addition, we have shown (Paper I) that the rate of translocation is adversely affected by an increased free concentration of magnesium ions.

Bacterial protein synthesis Bacterial protein synthesis can be divided into four phases, initiation, elon-gation, termination and recycling. During each phase a distinct set of transla-tion factors assists the ribosome in the assembly of a 70S initiation complex, in the synthesis of the nascent peptide chain, in peptide release or in disas-sembly of the ribosome into its subunits. The four phases will be described briefly in this section to give an overview of bacterial protein synthesis. In later sections the translocation step, which occurs during the elongation phase, and the ribosome recycling step, both of which involve the translation factor elongation factor G (EF-G), will be described in more detail.

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Initiation To initiate a new round of protein synthesis the IF3-bound 30S subunit, formed after splitting of the 70S ribosome into subunits during ribosome recycling, binds to an mRNA. The mRNA is positioned on the subunit by base-pairing of its SD sequence with the anti-SD sequence of the 16S rRNA. Initiation factor 1 (IF1) and the GTPase initiation factor 2 (IF2) bind to the 30S subunit and speed up the binding of initiator tRNA, fMet-tRNAfMet, to the start codon situated in the 30S P site (Figure 2). IF2 specifically recog-nizes the formylated methionine (15), which is unique for the initiator tRNA and not present in elongator Met-tRNAMet which reads the same codon. Up-on tRNA association to the 30S subunit the binding of IF3 is destabilized and the factor dissociates spontaneously (16). The destabilization is mutual and IF3 contributes to the accuracy of initiation by destabilizing the binding of all tRNAs to the 30S P site, both the initiator tRNA and elongator tRNAs (15). This enables rejection of incorrectly bound elongator tRNAs that would otherwise be trapped with high probability due to the very fast docking of the 50S subunit which occurs after IF3 dissociation. After 50S association, IF2 hydrolyses GTP which leads to dissociation of IF1 and IF2 from the ribosome (17).

Figure 2. Initiation of protein synthesis . A. mRNA binding to the IF3-bound 30S subunit. B. Binding of IF1, IF2(GTP) and fMet-tRNAfMet. C. Release of IF3 fol-lowed by 50S subunit association. D. GTP hydrolysis by IF2 and release of IF1 and IF2(GDP).

Elongation When initiation has been completed the ribosome enters the elongation phase, in which alternating peptide bond formation and translocation steps are repeated until the ribosome reaches a stop codon at the 3’-end of the mRNA (Figure 3). The 70S initiation complex, which contains an mRNA and an fMet-tRNAfMet in the P site, is ready to accept a ternary complex con-sisting of an aminoacylated tRNA bound to EF-Tu(GTP). After binding of the ternary complex to the ribosome the tRNA is in the so called A/T state in which its anticodon base-pairs with the mRNA codon on the 30S subunit, but its body remains bound to EF-Tu and is not yet accommodated in the 50S subunit A site (18). At this stage the bases G530, A1492 and A1493 of the 16S rRNA flip out to monitor the geometry of the two first base-pairs of the codon-anticodon helix and, if base-pairing is correct, they form stable

14

interactions with the minor groove of the short helix (19). If the base-pairing is not correct the ternary complex is rejected from the ribosome with high probability. How well the ribosome detects an error in this initial selection step varies greatly depending on which nucleotides are involved in the mis-match and whether it occurs in the first, second or third position of the co-don-anticodon helix (13, 20). Next, EF-Tu hydrolyses GTP and leaves the ribosome enabling accommodation of the aminoacyl-tRNA into the riboso-mal A site. Off the ribosome EF-Tu, assisted by elongation factor Ts (EF-Ts), exchanges its GDP for a new GTP molecule. Recent work from the Ehrenberg group (Ieong et al., unpublished) suggests that there are two proofreading steps, in addition to the initial selection step, that contribute to the overall accuracy of tRNA selection on the ribosome. The first step is the rejection of incorrect tRNAs in complex with EF-Tu(GDP) right after GTP hydrolysis and the second rejection step occurs after EF-Tu(GDP) dissocia-tion and tRNA accommodation in the ribosomal A site. Once the tRNA has been accommodated, the α-amino group of the amino acid is in position to make a nucleophilic attack on the carbonyl carbon of the ester linkage con-necting the nascent peptide to the P-site tRNA. The formation of a peptide (amide) bond results in breakage of the ester bond, deacylation of the P-site tRNA and transfer of the peptide, now elongated by one amino acid, to the A-site tRNA. Deacylation of the P-site tRNA enables ribosome ratcheting (21), which means rotation of the ribosomal subunits by about 6° in relation to each other (22), and formation of tRNA hybrid (A/P and P/E) states. In the hybrid states the ASLs of the two tRNAs remain bound to the 30S A and P sites but their acceptor ends enter the 50S P and E sites, respectively. Binding of EF-G in the GTP form to this complex stabilizes the ratcheted ribosome conformation as well as the tRNA hybrid states (23, 24). GTP hy-drolysis by EF-G precedes (25) the movement of the mRNA by one codon in

Figure 3. Translation elongation. A. Ternary complex binding. B. GTP hydrolysis by EF-Tu, release of EF-Tu(GDP) and tRNA accommodation. C. Peptide bond formation, tRNA hybrid state formation and ribosome ratcheting. D. EF-G(GTP) binding. E. GTP hydrolysis by EF-G, mRNA movement, displacement of the tRNAs into the P and E sites and ribosome back-ratcheting. F. Release of EF-G(GDP) and the E-site tRNA.

15

relation to the ribosome, displacement of the tRNAs into the P/P and E/E states and back-ratcheting of the ribosome. EF-G(GDP) is then released, which makes the ribosomal A site available for binding of another ternary complex.

Termination When the ribosome encounters a stop codon, a class I release factor (RF), RF1 or RF2, binds in the ribosomal A site and catalyzes the hydrolysis of the ester bond between the newly synthesized protein and the P-site tRNA (Figure 4). The two class I release factors have different specificities for the stop codons, UAG is recognized by RF1, UGA by RF2 and UAA by both factors (26). Subsequently the class I release factor is removed from the ri-bosome by the class II release factor, RF3 (27). RF3 either binds to the ribo-some in its GTP-bound form (28) or exchanges GDP for GTP after binding (29, 30). RF3(GTP) induces conformational changes in the ribosome, that lead to dissociation of the class I release factor (28, 31). RF3 hydrolyses GTP and leaves the post-termination ribosome, which contains an mRNA and a deacylated P site tRNA (29, 30).

Figure 4. Termination of protein synthesis . A. RF1 or RF2 binding and hydrolysis of the ester linkage between the P-site tRNA and the synthesized protein. B. RF3(GDP) binding. C. GDP to GTP exchange on RF3. D. RF1 or RF2 dissociation. E. GTP hydrolysis by RF3 and RF3(GDP) dissociation.

Recycling The ribosomal post-termination complex has to be disassembled to free the ribosomal subunits for initiation of protein synthesis on another mRNA tem-plate. This is achieved by EF-G together with ribosome recycling factor (RRF) (32) in a GTP hydrolysis-dependent reaction (33-36) (Figure 5). There are two opposing models for the sequence of steps in ribosome recy-cling. According to the model with the greatest amount of experimental sup-port (33), the first step is the release of the 50S subunit from the 70S post-termination complex by action of EF-G and RRF. In paper III we studied

16

this process in detail and determined a complete kinetic model for how the two factors achieve separation of the ribosomal subunits. IF3 then binds to the 30S subunit to catalyze the release of the deacylated tRNA bound in the P site (33, 34) and remains bound to prevent the subunits from reassociating. After dissociation of the tRNA the mRNA is free to dissociate from the 30S subunit.

Figure 5. Ribosome recycling. A. EF-G(GTP) and RRF binding. B. GTP hydrolysis by EF-G, 50S subunit dissociation, EF-G(GDP) and RRF dissociation (for the com-plete mechanism of steps A and B see Figure 10). C. IF3 binding to the 30S subunit followed by tRNA and mRNA release.

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The present work

This thesis deals with two of the phases in bacterial protein synthesis, elongation and ribosome recycling, both of which involve the action of the GTPase EF-G. During the elongation phase EF-G catalyzes the movement, or translocation, of the ribosome along the mRNA after incorporation of each amino acid. In paper I we describe the development of an assay for determining the average time of a complete translocation event including all steps from EF-G(GTP) binding to EF-G(GDP) release. We used this assay to study contextual effects, such as ribo-some position along the mRNA, magnesium ion concentration in the buffer and mRNA sequence, on the translocation rate. In paper III we determined the com-plete kinetic mechanism of the ribosome recycling reaction and demonstrated that RRF has to bind before EF-G to achieve ribosome splitting. We showed that the efficiency of the splitting reaction in terms of rate and GTP consumption varies greatly with the concentrations of the two factors.

The antibiotic fusidic acid (FA) binds to EF-G only when the factor is ribosome-bound and inhibits its activity in both translocation and ribosome recycling (37). In paper II we determined the mechanism of inhibition of elongating ribosomes by FA and showed that the drug targets three different ribosomal states during each elongation cycle. In paper IV we extended our mechanism for ribosome recycling from paper III to include inhibition by FA. We found that FA targets ribosome-bound EF-G both with and without RRF present in the post-termination complex. The overall strength of inhibition in recycling was similar to the effect on elongation, posing the interesting question of which of these two effects is most important in the living cell, upon treatment of a bacterial infec-tion with FA.

The common theme in the papers presented in this thesis is the use of fast kinet-ics methods, quench-flow and stopped-flow techniques, to study bacterial pro-tein synthesis in an in vitro translation system optimized for in vivo-like func-tion. We built kinetic models of EF-G activity and its inhibition by FA in trans-location and in ribosome recycling. We used mean time calculations for model evaluation and for obtaining precise estimates of the rate and equilibrium con-stants involved. In this section the experimental methods will be described, in-cluding the in vitro translation system, the quench-flow and the stopped-flow techniques as well as the mean time method for analysis of experimental data.

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The in vitro translation system All experiments were performed in an in vitro translation system containing purified components from the translational machinery of E. coli. All compo-nents, the ribosomes, translation factors, tRNAs, aminoacyl-tRNA synthetases etc., were purified individually and added separately to the reaction mixture. This makes the system versatile and adaptable for addressing a wide range of questions regarding ribosome function. The foundation of our translation sys-tem was the development of a buffer system optimal for high yield and accu-racy in synthesis of poly-phenylalanine peptides by ribosomes programmed with poly-U mRNA (12). The resulting polymix buffer contained naturally occurring cations (magnesium, calcium and potassium) as well as the polyam-ines spermidine and putrescine, which are also present in the cell. The total magnesium ion concentration in the polymix buffer is 5 mM, but addition of compounds needed for energy supply in the system, ATP, GTP and phosphoe-nol pyruvate (PEP) (1 mM, 1 mM and 10 mM, respectively at standard condi-tions), which all chelate magnesium ions reduces the free Mg2+ concentration to around 1.3 mM (13). This is very low compared to what is commonly used in biochemical experiments aimed at studying ribosome function, but close to the free magnesium ion concentration inside an E. coli cell, which is around 1 mM (38). Our in vitro translation system has to date been used for detailed studies of most of the different translational phases and has shown high and in vivo compatible rates of initiation (16), peptide bond formation (39), transloca-tion (Paper I), termination (40) and ribosome recycling (Paper III) as well as high accuracy of tRNA selection (13).

The quench-flow technique Studies of ribosome function under in vivo-like conditions often require the application of experimental techniques developed for studying pre-steady state kinetics. In a quench-flow instrument two solutions containing the reac-tants are rapidly mixed in a reaction loop, the reaction mixture is incubated until it reaches the outlet of the reaction loop where it is quenched by mixing with a quencher, in my case usually formic acid (Figure 6). The quench-flow technique can be used for studying reactions resulting in permanent changes detectable in the quenched samples, usually involving breakage or formation of covalent chemical bonds. In the work presented here we used it to study the formation of peptide bonds in short peptides containing a radioactively labeled fMet residue and the hydrolysis of radiolabeled GTP to GDP, both of which could be analyzed by HPLC separation with radioactivity detection. In the assay that we developed for studying translocation deep inside an open reading frame (Paper I), radiolabeled amino acids were incorporated at the end of a peptide. These long peptides could not, due to poor solubility, be

19

analyzed by HPLC but were isolated by nickel affinity chromatography and amino acid incorporation was quantified by scintillation counting.

Figure 6. Schematic representation of a quench-flow instrument. The three syringes at the top of the device are simultaneously pushed down by a motor. This pushes the reactant mixtures (A and B) from the sample loops into the reaction loop where they rapidly mix. At the end of the reaction loop the sample is mixed with a quencher, which stops the reaction. The samples are collected through the exit loop.

The stopped-flow technique and Rayleigh light scattering The stopped-flow instrument has a similar setup as the quench-flow instru-ment, the reactants from two syringes are rapidly mixed, but the reaction is not quenched and instead monitored in real time. The reaction mixture is held in a reaction cell on which light is shone and changes in for example absorption, fluorescence or light scattering intensity are recorded, depending on the reaction studied. In paper II the movement of mRNA through the ribosome upon translocation was monitored as a decrease in fluorescence from a pyrene label added to its 3’-end according to the method of Studer et al. (41). In papers III and IV splitting of the post-termination ribosomal complex was monitored as a decrease in Rayleigh light scattering intensity (42). This method uses the fact that the incident light induces coordinated oscillations of the electrons in the particles in the sample with periodic charge separations (dipole moments) resulting in emission (scattering) of light in all directions (43). The light scattering intensity is proportional to the square of the molecular weights of the particles, the size of which has to be

20

much smaller than the wavelength of the incident light. Association or disso-ciation of two molecules results in a relative change in the light scattering intensity which is largest when the two particles have equal molecular weights. Figure 7 shows a time trace from an RRF and EF-G catalyzed ribo-some splitting reaction recorded in the stopped-flow instrument (Paper III).

Figure 7. Time trace of ribosome splitting recorded in the stopped-flow instrument, upper trace. The lower trace is the background light scattering recorded in the ab-sence of ribosomes.

A useful aspect of the light scattering technique is that the fraction of split ribosomes can be calculated from the light scattering intensity change. The intensity at the start of the reaction, when all ribosomes are in the 70S form, is I(0) and it then decreases gradually as splitting occurs, eventually reaching a plateau value, I(∞). Background light scattering, I(bg), originating from the buffer and other components in the reaction mixture, was recorded in paral-lel under the same conditions but in the absence of ribosomes. Since the light scattering intensity is proportional to the square of the molecular weights of the particles the maximal relative intensity change that would occur if all ribosomes were split is given by

( )( )

( )

( )( )

2 2 230 50 30 50

230 50

30 502

30 50

(max) (0) ( )

2 (0) ( )

S S S S

S S

S S

S S

M M M MI I I bg

M MM M I I bg

M M

+ − −∆ = −

+

= −+

(i)

The observed light scattering intensity change for the ribosome splitting reaction is I(0)-I(∞) and the fraction of split ribosomes is thus given by, as exemplified by the reaction in Figure 7:

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( )230 50

30 50

(0) ( ) 7.7 6.3 0.84(0) ( ) 2 0.462 (7.7 4.1)

S Ssplit

S S

M MI IPI I bg M M

+− ∞ −= = =

− ⋅ − (ii)

Here, the molecular weights of the ribosomal subunits are set to 0.85 and 1.5 MDa, respectively (44). In this case 84% of the ribosomes were split in the reaction. In papers III and IV we determined the amount of split ribosomes as a function of time in the stopped-flow instrument, as described here, and in parallel we determined the amount of GTP consumed by the reaction us-ing the quench-flow instrument. Taking the ratio of these two, we were able to determine the number of GTP molecules consumed per ribosome splitting event at varying concentrations of EF-G and RRF.

Mean time calculations In all papers included in this thesis we have used mean time calculations for evaluating data from biochemical experiments in terms of more or less com-plex kinetic models. Any kinetic scheme can be converted into a set of dif-ferential equations that describes how the probability of being in the differ-ent states varies with time. A simple mechanism of two consecutive irre-versible steps (iii) may serve as an illustrative example.

A Bk kA B C→ → (iii)

For this scheme we get the following differential equation system:

( ) ( )

( ) ( ) ( )

AA A

BA A B B

dP t k P tdt

dP t k P t k P tdt

= − = −

(iv)

Here PA(t) and PB(t) are the time-dependent probabilities that a molecule is in state A or B, respectively. The probability of being in any state, X, at any time point is the probability of having entered the state before that time mi-nus the probability of having left the state:

( ) ( ) ( )X in outP t P t P t= − (v)

Pin(t) and Pout(t) are cumulative distribution functions that increase from zero to one with time and Pin(t) is at all times larger than Pout(t). The probability of leaving state X per unit time is thus:

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( )( ) ( )outX indP tdP t dP tdt dt dt

− = − (vi)

The average time (τX) that is spent in state X can be calculated as the average time for leaving the state minus the average time for entering the state, which are given by the expectation values of the probability density func-tions dPin(t)/dt and dPout(t)/dt:

[ ]

0 0

0Partial integration0 0 0

( ) ( )

( ) ( ) ( ) ( )

out inX out in out in

XX X X

dP t dP tt t t dt t dtdt dt

dP tt dt t P t P t dt P t dtdt

∞ ∞

∞ ∞ ∞∞

t = t − t =< > − < >= − =

− = − ⋅ + =

∫ ∫

∫ ∫ ∫ (vii)

The term t·PX(t) is zero both when t is zero and when t goes to infinity, since PX(t) decreases exponentially and thus faster than t increases. We see that τX, can be obtained by simply integrating the time-dependent probability of be-ing in state X, PX(t), from zero to infinite time (Figure 8a and b). Hence, if the differential equation system (iv) is integrated from zero to infinite time an algebraic equation system for the average times spent by the system in each intermediate state is obtained.

0 0

0 0 0

( ) ( )

( ) ( ) ( )

AA A

BA A B B

dP t dt k P t dtdt

dP t dt k P t dt k P t dtdt

∞ ∞

∞ ∞ ∞

= −

⇒ = −

∫ ∫

∫ ∫ ∫ (viii)

(0)(0)

A A A

B A A B B

P kP k k− = − t− = t − t

(ix)

The initial conditions, PX(0), describe the reaction system at time zero. For example, if the system starts in A, we set PA(0) = 1, PB(0) = 0 and PC(0) = 0, and solve the algebraic equation system (ix) to obtain the average times spent in states A and B as τA = 1/kA and τB = 1/kB. Thus, for an irreversible reaction step the average time spent in the preceding state is simply the in-verse of the rate constant for leaving the state.

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Figure 8. (a) Time evolution of the probabilities of being in the different states A through C for the mechanism of two irreversible steps (iii), with kA = 30 s-1 and kB = 10 s-1. The grey-shaded area under the curve PB(t) corresponds to the average time that the system spends in state B, τB = 1/kB = 100 ms. (b) Time-dependent probabili-ties of entering Pin,B(t) = PB(t) + PC(t) and leaving, Pout,B(t) = PC(t) state B. The prob-ability of being in state B is PB(t) = Pin,B(t) - Pout,B(t) and hence the area between the curves corresponds to τB. The expectation values of dPin,B(t)/dt and dPout,B(t)/dt are the average times for entering and leaving state B, respectively (τin,B = 33 ms τout,B = 133 ms) and the difference between these two times is the average time spent in state B, τB = 100 ms (vii). (c) Curves as in (a), the shaded area corresponds to the average total time for reaching the end state (C) of the reaction (iii), τtot = τA + τB = (1/30 + 1/10) s = 133 ms.

The total average time (τtot) required for reaching the end state of the reaction (state C in the example) is the sum of the average times spent in all preced-ing states.

( ) ( )0 0

( ) ( ) 1 ( )tot A B A B CP t P t dt P t dt∞ ∞

t = t + t = + = −∫ ∫ (x)

Eq. (x) shows that the total time for completing the reaction is given by the integral of the area between P(t) = 1 and the curve describing accumulation of the end product as illustrated in Figure 8c. Hence, without knowing any-thing about the underlying kinetics of the reaction leading to product for-mation the total reaction time can always be determined simply by integrat-ing the area over the curve.

It is of course more useful if the observed reaction kinetics can be connected to a kinetic scheme describing the reaction. For reaction mechanisms with reversible reaction steps or branched reaction pathways the total time ex-pression becomes much more complex than in the simple example described here (Papers II, III and IV). The total time expression describes how the average time of product formation varies with the reactant concentrations, in my case translation factors or the antibiotic drug fusidic acid. By examining the expression derived from a proposed reaction model, predictions can be made about the behavior of the system and experiments can be designed for

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critical testing of the model. From this type of analysis it is usually clear whether a model is sufficient for explaining the observed variations in the data or if a more complex model is needed. If the model is sufficient for explaining the experimental observations and the data is of good quality, fitting of the total time expression yields high precision estimates of some or all of the rate constants in the kinetic scheme, depending on the complexity of the system.

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EF-G and translocation

The task of EF-G during each elongation cycle is to translocate the tRNAs bound in the ribosomal A and P sites into the P and E sites and at the same time move the mRNA by one codon.

The pre-translocation complex The substrate for EF-G binding is the ribosomal pre-translocation complex in which the nascent peptide, as a result of peptide bond formation, has been extended by one amino acid and transferred from the P- to the A-site tRNA. The coincident deacylation of the P-site tRNA is the key event that unlocks the ribosome and allows it to enter the ratcheted state, which is efficiently targeted by EF-G(GTP) (21, 45), and enables the tRNAs to enter their hybrid binding states. As was first observed in chemical footprinting experiments performed by Moazed and Noller (46), the acceptor (CCA) ends of the A- and P-site tRNAs move in relation to the 50S subunit to base-pair with the 23S rRNA in the P and E sites, respectively. This movement, which occurs spontaneously after peptide bond formation, was thought to be coupled to the rotational movement of the ribosomal subunits.

Recent single molecule Förster resonance energy transfer (FRET) methods have made it possible to analyze tRNA binding states as well as ribosome dynamics in detail. Such experiments rely on the detection of distance-dependent energy transfer between fluorescent probes attached to tRNAs or ribosomal proteins. The pre-translocation ribosome was shown to undergo spontaneous transitions between the ratcheted and non-ratcheted confor-mations (47) and the tRNAs in this complex displayed spontaneous transi-tions between the classical (A/A and P/P) and hybrid (A/P and P/E) binding states (48, 49). One study suggested tight coupling between ribosome ratch-eting, tRNA hybrid state formation and L1 stalk closing (50) so that the pre-translocation complex would fluctuate between two global states, the first non-ratcheted with the tRNAs in their classical states and the L1 stalk in an open conformation and the second ratcheted with the tRNAs in hybrid states and the L1 stalk closed. In the closed conformation the L1 stalk, which con-sists of helices 76-78 of 23S rRNA and ribosomal protein L1, moves inward towards the intersubunit space and interacts with the tRNA in the P/E state.

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The notion of two global states was supported by visualization of the pre-translocation complex by cryo-EM (51, 52), but has remained controversial (53, 54).

The ribosome and tRNA dynamics in the pre-translocation complex, ob-served in single molecule experiments, are generally very slow with transi-tion times ranging from about 100 ms up to many seconds (47-49, 55). This slowness can partially be attributed to low reaction temperatures (20-25 °C), but the relevance of the results in relation to the in vivo situation, where a complete elongation cycle takes only about 50 to 100 ms at 37 °C (1), re-mains questionable. Contrasting previous observations, a recent study, per-formed at a comparably low and in vivo like (38) free Mg2+ concentration (1 mM), showed no ratcheting transitions in the pre-translocation ribosome (56). Under these conditions the ribosome was found to reside mainly in the ratcheted state, which agrees well with the previously observed increasing propensity for tRNAs to occupy the hybrid state with decreasing magnesium ion concentration (49, 51, 52). However, in an earlier study lowered magne-sium had been shown to increase the transition rates between the classical and hybrid tRNA binding states (49). All these observations taken together, point to that the pre-translocation ribosome in vivo is mainly found in the ratcheted state with the tRNAs in the hybrid states. However, the possible interconnection between ribosome ratcheting and tRNA dynamics and whether they play a practical role in vivo remains unclear.

GTP hydrolysis and Pi release Binding of EF-G in complex with a non-hydrolyzable GTP analogue, mim-icking its GTP-bound state, to the pre-translocation ribosome stabilizes the ratcheted state (21, 24, 50). GTP hydrolysis by EF-G leads to conformational changes in the factor, back-ratcheting of the ribosome and translocation of the mRNA and the two tRNAs in relation to the 30S subunit. Despite con-siderable efforts it remains largely unknown how the energy from GTP hy-drolysis is converted into ribosomal motion. EF-G is able to bind to many different types of ribosomal complexes, although with varying efficiency, reflected by the greatly varying kcat/KM-values for GTP hydrolysis by the factor at different types of complexes. We have observed binding of EF-G to pre-translocation complexes with a peptidyl-tRNA in the A site and a deac-ylated tRNA in the P site (Paper I and II), to initiation complexes with fMet-tRNAfMet in the P site (Paper II), to post-termination complexes with a deac-ylated tRNA in the P site with and without RRF (Paper III) and to free 50S subunits (Paper III). In all cases ribosome binding stimulates GTP hydrolysis (25), but it is only for certain specific complexes that the released energy is converted into useful work, such as translocation or splitting of the RRF-

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bound post-termination complex. The promiscuity of EF-G in targeting vari-ous ribosomal states complicates biochemical studies of the factor, since background reactions are often difficult to separate from the signal of inter-est. Intimately connected to GTP hydrolysis is the release of inorganic phos-phate (Pi) from EF-G(GDP·Pi) formed in the reaction. Whether this event plays an active role in promoting translocation or if it is just an unavoidable consequence of GTP hydrolysis remains unclear. Pointing to the latter, it was suggested that Pi release occurs independently of the translocation movement of tRNAs and mRNA (57), but further experimentation is needed to resolve this question.

Structural studies of EF-G in translocation With the aim of visualizing the conformational states of EF-G and of the ribosome that occur during translocation, many high resolution structures of EF-G bound to the ribosome with non-hydrolyzable GTP analogues and various antibiotics have been determined. In many cases the complexes con-tained a single deacylated tRNA in the P site and did therefore not, due to the lack of an A-site tRNA, represent true translocation intermediates. In most of these structures Domain IV of EF-G was bound in the 30S A site, incompatible with tRNA binding to this site. The complexes display a vary-ing degree of ribosome ratcheting as well as varying extent of swiveling (rotation) of the 30S subunit head domain. In some structures, where the tRNA was bound in the P/E state, reminiscent of a pre-translocation ribo-some, the subunit rotation was about 7° and the head swivel 5 to 9° (58-60). In other structures the ASL of the tRNA had moved partially towards the E site on the 30S subunit, into a so called pe/E state. These structures displayed only 3 to 5° ratcheting but a large head swivel of about 15 to 18° (60, 61) suggesting that back-ratcheting of the ribosome and concomitant forward rotation of the 30S subunit head, the latter interacting with the P-site tRNA, facilitates tRNA and mRNA movement towards the E site.

More recently, structures of EF-G bound to ribosome complexes containing an aminoacyl-tRNA or a peptidyl-tRNA analogue in the A site and a deacyl-ated tRNA in the P site have been determined. In two of these structures the tRNAs were found in intermediate states of translocation, in which their ASLs had moved partially in relation to the 30S subunit to positions between the 30S P and E sites and the 30S A and P sites, respectively (62, 63). These structures display a partially back-ratcheted state with about 3° subunit rota-tion and large (18° and 21°, respectively) head swiveling, in agreement with previous observations. Domain IV of EF-G was found to interact with the codon-anticodon helix of the tRNA bound between the A and P sites on the 30S subunit. The same interaction was observed also in an EF-G- and vio-

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mycin-bound pre-translocation ribosome with tRNAs in the A/P and P/E states, visualized by cryo-EM (64) and in a crystal structure of a post-translocation-like complex with EF-G stabilized by fusidic acid on a non-ratcheted ribosome with deacylated tRNAs in the P/P and E/E states (65). Therefore the interaction of domain IV with the codon-anticodon helix is likely maintained throughout the translocation step and is thought to facili-tate the tRNA movement from the A to the P site. In addition, all four struc-tures displayed interaction of the L1 stalk with the tRNA that proceeds from the P/E state to the E/E state, in agreement with results from a single mole-cule study (50). An intermediate L1 stalk conformation, between the open and closed, was demonstrated for the post-translocation state, in which it interacts with the E-site tRNA (66). Complete opening of the L1 stalk after translocation has been suggested to facilitate tRNA release from the E site (21).

Biochemical studies of EF-G in translocation In paper I we developed an assay for determining the complete translocation time at any position in any open reading frame. The translocation time measured by this method includes all EF-G-dependent steps; EF-G(GTP) binding, GTP hydrolysis, Pi release, tRNA and mRNA movement, ribosome back-ratcheting and EF-G(GDP) release. We exploited that the ribosome could be stalled in the post-translocation state on the first occurrence of any amino acid in the peptide sequence by omitting that amino acid and the cor-responding aminoacyl-tRNA synthetase from the reaction mixture. Elonga-tion was then resumed by supplying the missing ternary complex for the A-site codon and the incorporation of the next two amino acids was followed over time. The average total time of all steps leading to incorporation of the second amino acid (τtot) is the sum of the average times of two peptide bond formation steps (τp1 and τp2) and one intervening translocation step (τtrans) (Figure 9a). Time curves for the overall three-step reaction and for the for-mation of the first peptide bond were obtained starting from the same stalled complex, whereas the time curve for formation of the second peptide bond was obtained starting from a complex stalled one codon further downstream (Figure 9b). Simultaneous fitting of the three time curves yielded precise estimates of the average times of all three reaction steps.

We studied the first translocation step after initiation as well as a transloca-tion step fifteen codons downstream from the initiation site and determined the translocation rate (=1/τtrans) at varying concentrations of EF-G in the two cases (Figure 9c). We found that the maximal translocation rate (kcat) was about twofold faster at the downstream site (22 s-1 vs. 13 s-1) and that the EF-

29

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Figure 9. (a) Incorporation of two consecutive amino acids on a stalled post-translocation ribosomal complex. The first peptide bond formation step, with aver-age time τp1, is followed by translocation, with average time τtrans, and a second pep-tide bond formation step, with average time τp2. The overall three-step reaction has the average time τtot = τp1 + τtrans + τp2. (b) Time courses of the first (yellow) and second (green) peptide bond formation steps and the overall three-step reaction (blue) obtained at 1.5 µM of each ternary complex and 0.5 µM EF-G. Fitting to the three-step model described in (a) yielded estimates of the average times as τtrans = 124 ± 8 ms, τp1 = 32 ± 2 ms and τp2 = 25 ± 1 ms. (c) EF-G concentration dependence of the translocation rate (1/τtrans) for the first translocation step (short ORF) and a translocation step 15 codons downstream from the initiation site (long ORF). Given on the x-axis is the total concentration of EF-G as determined by the Bradford assay, out of which 51% is active.

G concentration required to reach half of this maximal rate (KM) was about half (0.65 µM vs. 1.25 µM, values adjusted compared to the ones given in paper I for an EF-G activity of 51% determined in paper II). This result is in agreement with previous studies showing lower propensity of tRNA hybrid state formation in the pre-translocation ribosome and slower translocation of fluorescence labeled mRNA through the ribosome (67, 68) with a deacylated initiator tRNAfMet in the P site, than with an elongator tRNA (47, 69). The presence of a peptidyl-tRNA in the A site rather than an aminoacyl-tRNA also stimulates hybrid state formation (48, 50), suggesting that the length of the peptide may also play a role in promoting translocation. The maximal rate of 22 s-1 that we obtained for the complete translocation process is fast

enough to be compatible with in vivo elongation rates of about 15 to 20 ami-no acids per second (70-72). Even faster translocation rates of about 35 s-1 were observed on mRNA templates with a different coding sequence preced-ing the translocation site, indicating a possible influence of the mRNA or peptide sequence on the rate of translocation.

Previous assays have targeted only sub-steps in translocation but not the complete process. One common assay makes use of the antibiotic puromy-cin, which is an analogue of the aminoacylated 3’-end of a tRNA. Puromy-cin binds in the 50S A site and participates in peptide bond formation just like an aminoacyl-tRNA, but then leaves the ribosome together with the peptide, causing premature termination. Puromycin reacts poorly with a pep-tidyl-tRNA in the hybrid A/P state but has increased reactivity towards tRNA in the P/P state. However, exactly at what stage during translocation that the increased reactivity occurs is not known. When the native reaction, including ternary complex binding and regular peptide bond formation, is used to probe translocation, as in our assay, there is no doubt that transloca-tion has been completed and that EF-G has dissociated before reactivity oc-curs.

Ribosome ratcheting has been studied with FRET-based methods in bulk (23) as well as in single molecule experiments (47, 56), as described above. Fluorescence-based methods are commonly used to follow the progress of tRNAs and mRNA through the ribosome. Joseph and coworkers attached a fluorescent pyrene label to the 3 -́end of the mRNA to monitor its movement during translocation (41). Decreased fluorescence intensity was observed as the pyrene label was pulled into the ribosome. We used this assay in paper II to show that the rate of mRNA movement remained unchanged in the pres-ence of fusidic acid. Apart from the risk that the attachment of fluorescent probes may disturb the reaction under study, results from fluorescence based assays are often difficult to interpret since the molecular event causing the fluorescence intensity change is usually not known. For example, the py-rene-based assay (41) has recently been speculated to monitor the back-rotation of the 30S subunit head, rather than the mRNA movement (73), although this would need to be confirmed by further experimentation. In another study two different Alexa dyes that were used in place of pyrene for monitoring mRNA movement displayed a fivefold difference in transloca-tion rate although attached to the same position of the mRNA (74), suppos-edly reflecting two different molecular events. Proflavin is commonly used for fluorescence labeling of tRNAs (75). Further illustrating the ambiguity of fluorescence based methods, attachment of this dye to two different positions on the A-site tRNA gave rise to very different signals upon translocation, single exponential in one case and biphasic in the other (76).

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We used our translocation assay to study the effect of magnesium ions on the rate of translocation. The maximal translocation rate (kcat) decreased from about 30 s-1 at 1 mM free Mg2+ to 1 s-1 at 6 mM free Mg2+. At the same time, the EF-G concentration required to reach half of this maximal rate (KM) de-creased from about 1 µM at 1 mM Mg2+ to a value smaller than 0.25 µM at 6 mM Mg2+ (values adjusted for an EF-G activity of 51%). This resulted in a roughly constant kcat/KM-value of about 30 µM-1s-1 for the binding of EF-G to the pre-translocation complex at all Mg2+ concentrations. Translocation inhibition by magnesium ions has many possible explanations. Stabilization of inter-subunit bridges in the non-ratcheted state may result in a larger en-ergy barrier for the ratcheting movement, reflected by higher classical state occupancy at increasing magnesium ion concentrations (49, 51, 52). Magne-sium ions have been suggested to increase the affinity of tRNA binding to the ribosomal E site (51), which may also interfere with ratcheting and cause slower translocation. In an early cryo-EM study (21) the pre-translocation ribosome was found exclusively in the non-ratcheted state, which might be explained by the presence of an E-site tRNA in this structure. We suggested in paper I that magnesium ions may also inhibit translocation by stabilizing the flipped out conformation adopted by the monitoring bases, G530, A1492 and A1493 in 23S rRNA, for monitoring of correct base pairing in the co-don-anticodon helix during decoding (19). This would counteract their dis-placement which is likely induced by EF-G at an early stage in the transloca-tion process. This hypothesis was based on the assumption that magnesium ions and aminoglycosides, both of which decrease the accuracy of tRNA selection in the ribosomal A site as well as the rate of translocation do so by the same mechanism. However, recent work from the Ehrenberg group (Zhang et al., unpublished) suggests that whereas aminoglycosides stabilize the codon-specific binding of a cognate ternary complex, magnesium ions increase the non-specific binding of all ternary complexes to the ribosomal A site. It is possible that magnesium ions inhibit translocation by increasing the affinity of the tRNA for the ribosomal A site, impeding its movement to-wards the P site. High magnesium ion concentrations are commonly used in biochemical studies of ribosomal protein synthesis. Our results clearly show that ribosome function is greatly impaired under these conditions, with a thirtyfold reduction in the translocation rate as the free Mg2+ concentration increases from 1 to 6 mM to rates incompatible with in vivo elongation rates.

It has been suggested from ribosome profiling experiments, in which the distribution of ribosomes on all mRNAs inside living cells is analyzed, that the rate of elongation is determined by interactions between SD-like se-quences in the mRNA, situated upstream of the codon, and the anti-SD se-quence in the 16S rRNA. A strong interaction leads to slow elongation, ob-served as a higher occupancy of ribosomes at that codon, tentatively due to a reduction in the translocation rate. We used our translocation assay to test

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this hypothesis and constructed a set of mRNA templates containing SD-like sequences, with different affinities for the antiSD sequence, upstream of the translocation site. We did not observe any variation in the translocation rate in our small set of mRNAs, but the effect might be seen if a more extensive study is undertaken.

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EF-G and ribosome recycling

There are two opposing models for the sequence of events during ribosome recycling. In the first model, the deacylated P-site tRNA is released from the post-termination complex at an early stage upon a translocation-like move-ment of RRF through the ribosome catalyzed by EF-G (36). The idea of a translocation-like mechanism originated from the similarity in shape be-tween RRF and a tRNA (77). However, it has been shown that RRF binds to the ribosome in a different orientation than the tRNAs (78-81), so a possible translocation of RRF must be achieved in a different way. Furthermore, the model suggests that, after tRNA release, RRF and EF-G transiently split the ribosome into subunits, enabling mRNA release. To keep the subunits stably separated IF3 binds to the 30S subunit. In the second model, the 50S subunit is released first, by action of EF-G and RRF (33, 35). The mechanism of this step, which does not require the involvement of IF3 (82), was investigated in great detail in paper II. After ribosome splitting IF3 binds to the 30S subunit to keep it from rebinding to the 50S subunit, but also to catalyze the release of the tRNA (33). Once the tRNA has been removed, the mRNA spontane-ously dissociates from the 30S subunit.

There is a large amount of structural data available on RRF-bound ribosome complexes. In crystallographic studies RRF was soaked into preformed crys-tals of vacant ribosomes (83, 84), of 50S subunits (85) or of ribosomes with mRNA and an ASL fragment in the P site (86). There is also a cryo-EM structure of RRF bound to a vacant ribosome (87). These studies revealed that RRF interacts with both the A and P sites of the 50S subunit, thereby blocking tRNA binding to these sites. RRF was seen to interact with helix 69 (H69) which forms an important inter-subunit bridge (B2a) together with helix 44 (h44) of the 30S subunit. The difference in composition of these complexes from a post-termination complex and the immobility of the ribo-some within preformed crystals reduce the functional relevance of these structures. More relevant structures, determined by low resolution cryo-EM (79, 80) and high resolution X-ray crystallography (81), show RRF bound to the intact post-termination complex, with mRNA and a deacylated P-site tRNA. In these structures the ribosome was in the ratcheted conformation and the tRNA in the P/E hybrid state. Domain I of RRF was bound close to the subunit interface between helices 69 and 71 in the A and P sites of the 50S subunit (79, 81). Domain II is allowed more flexiblity and has been

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found in several different conformations. The high resolution crystal struc-ture showed that the interactions between H69 and H71 with h44, in the in-ter-subunit bridges B2a and B3, respectively, remained intact also in the ratcheted state, although H69 had a compressed conformation (81).

Based on a comparison between the conformation of RRF in the 70S post-termination complex and in a 50S·EF-G(GDPNP)·RRF complex it was sug-gested that a large movement of RRF domain II induced by EF-G may be disrupting inter-subunit bridges B2a and B3 and thus cause separation of the ribosomal subunits (79). In the 50S structure domains III and V of EF-G interacted with the hinge region between the two RRF domains and domain IV of EF-G interacted with domain II of RRF (79, 88). In this structure RRF domain II would overlap with h44 and protein S12 if the 30S subunit would be present. The orientation of RRF domain II in the two structures differs by as much as 60°, possibly due to steric or electrostatic repulsion by EF-G domain IV (88). Upon GTP hydrolysis domains III, IV and V of EF-G move towards the 30S subunit, as seen from comparison to a 50S·EF-G(GDP)·FA structure reflecting the post-hydrolysis state. This movement possibly induc-es the extensive rotation of RRF domain II (88).

The RRF- and EF-G-bound post-termination complex is, due to its short lifetime, difficult to visualize by structural methods. Shortly after binding of both factors to the ribosome, the complex will decompose, by separation of the two ribosomal subunits. A classical way to solve a problem like this is to assemble the complex using a non-hydrolyzable GTP analogue instead of GTP. However, forming a 70S complex with RRF, EF-G and a non-hydrolyzable GTP analogue has not been possible (88). Only one structure displaying EF-G and RRF bound simultaneously to the post-termination complex is available (89). This complex contains heterologous translation factors, RRF from T. thermophilus and EF-G from E. coli, a factor combina-tion inactive in ribosome splitting (90), and therefore the functional rele-vance of this structure is not clear.

In paper III we determined the complete kinetic mechanism of ribosome recycling (Figure 10) by EF-G and RRF. We showed that ribosome splitting can only be achieved if RRF binds before EF-G to the post-termination complex. The maximal rate of splitting, reached at very high concentrations of EF-G and RRF and a high RRF-to-EF-G concentration ratio was 25 s-1 at 37 °C. Binding of EF-G to the factor-free post-termination complex, in com-petition with RRF, resulted in inhibition of the recycling reaction and in wasteful hydrolysis of GTP. The extent of inhibition and of the related futile GTP hydrolysis was determined by the ratio of the RRF and EF-G concen-trations. An increased RRF concentration reduced the number of GTP mole-cules consumed per ribosome splitting event. At very high concentrations of

35

36

RRF the GTP consumption was determined by the ratio of the rate of release of EF-G(GDP) from the RRF- and EF-G-bound post-termination complex (qG2) and the rate of splitting of the same complex (ksplit). The rate of EF-G(GDP) release was low, giving efficient splitting of the post-termination complex after binding of RRF and EF-G, as reflected by the consumption of a single GTP molecule per ribosome splitting event at high concentrations of RRF.

Figure 10. The mechanism of ribosome recycling determined from our biochemical experiments. EF-G(GTP) binds to the post-termination complex with rate constant kG1. It may then either be released with rate constant qG(GTP)1 or hydrolyze GTP with rate constant kGTP1. After GTP hydrolysis EF-G(GDP) is released with rate constant qG1. EF-G binding to the post-termination complex competes with the binding of RRF, thus inhibiting the ribosome recycling reaction. RRF binds to the post-termination complex with rate constant kRRF and dissociates with rate constant qRRF. EF-G(GTP) binds to the RRF-bound post-termination complex with rate constant kG2 and may then either be released with rate constant qG(GTP)2 or hydrolyse GTP with rate constant kGTP2. The rate of release of EF-G(GDP) from the two-factor complex, qG2, is low and a major fraction of the RRF- and EF-G-bound post-termination complexes are split into subunits with rate constant ksplit.

EF-G and fusidic acid

Given the vital role of ribosomes in bacterial cells it is not surprising that they are the target of numerous antibiotics. Most ribosome-targeting drugs bind close to the functionally important regions, such as the A and P tRNA binding sites on the 30S subunit (e.g. tetracyclines, aminoglycosides and tuberactinomycins) or the peptidyl transferase center (e.g. chloramphenicol and puromycin) and the peptide exit tunnel (macrolides, ketolides) in the 50S subunit (91). There are a few examples of protein synthesis inhibitors that target translation factors, rather than the ribosome itself. Pulvomycin binds to free EF-Tu and prevents ternary complex formation and kirromycin binds to ribosome-bound EF-Tu during decoding and prevents its release from the ribosome (92). Similarly, fusidic acid (FA) binds to ribosome-bound EF-G during translocation and ribosome recycling (37). Many antibi-otics are used as molecular tools for studying ribosome function, but surpris-ingly little is known about their mechanisms of action at the molecular level. In paper II and IV we determined the complete kinetic mechanisms for FA inhibition of translocation (Figure 11) and ribosome recycling (Figure 12).

Fusidic acid is an antibiotic naturally produced by the fungus Fusidium coc-cineum (93) and used to fight infections in skin, eyes, soft tissue and bone caused by Gram-positive bacteria such as Staphylococcus aureus. FA inter-acts poorly with free EF-G, but has high affinity for the factor when it is ribosome-bound (94, 95). Two structures of EF-G bound with FA to ribo-somes in the translocation phase have thus far been presented. One, deter-mined by cryo-EM, displayed an intermediate translocation state, in which the mRNA and the tRNAs had moved partially in relation to the 30S subunit (63). The other, determined by X-ray crystallography, displayed a non-ratcheted post-translocation-like state of the ribosome (65). FA binds in a pocket between domains I, II and III of EF-G. Domain I, or the G-domain, contains the switch I and switch II loops that interact with the γ-phosphate of GTP (96). In structures of EF-G bound to the ribosome with a non-hydrolyzable GTP analogue, GMPPCP (97) or GDPNP (60), the switch re-gions are ordered and form a closed nucleotide binding pocket. When EF-G in the GDP form is trapped on the ribosome by FA, the switch I element is disordered (60, 65). The FA binding site overlaps with the ordered confor-mation of switch I, suggesting that the drug can only bind after GTP hydrol-ysis and disordering of the loop. The switch loops are believed to sense the

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phosphorylation state of the nucleotide, coupling it to conformational chang-es in EF-G during translocation. When EF-G is free in solution the switch loops are disordered (96) and stabilization of switch II by FA may prevent the conformational changes in EF-G necessary for completion of transloca-tion and subsequent dissociation of the factor from the ribosome (65).

Antibiotic resistance is an increasing problem in public health care. Several mechanisms of resistance against FA are known. Gram negative bacteria, like E. coli, are naturally protected by their impermeable outer membrane. Mutations in EF-G confer FA resistance either by weakening the drug bind-ing to EF-G or by altering the ribosome-EF-G interaction (98-100). Another mechanism is the expression of plasmid-encoded proteins conferring re-sistance to FA, for example FusB (101). FusB, which interacts with S. aure-us EF-G but not E. coli EF-G, alleviates inhibition of both translocation and ribosome recycling, possibly by promoting disassembly of FA-stalled ribo-some complexes (102, 103). Mutations that cause complete loss or trunca-tion of ribosomal protein L6 have also been shown to confer FA resistance, by altering the binding site for EF-G on the ribosome (104).

In paper II we analyzed tripeptide formation kinetics in the presence of FA. Time courses obtained at sub-saturating concentrations of the drug had a characteristic biphasic appearance. Each curve had a fast and a slow phase, reflecting tripeptide formation on FA-free and FA-bound ribosomes, with average times τA and τI, respectively. The fraction of ribosomes that were inhibited by FA was reflected by the fractional amplitude of the slow phase, pI, which increased from zero in the absence of FA to one at saturating FA concentrations. The slow phase average time, τI, increased linearly with the FA concentration, also after saturation, due to repeated FA binding events. The parameters τA, τI and pI were precisely estimated by simultaneous fitting of time traces obtained in the presence and absence of FA and used to calcu-late the total time of tripeptide formation, τtrip = τA + pIτI, for each trace. The total tripeptide formation time increased nonlinearly with the FA concentra-tion, proving that FA binds to more than one intermediate state during each elongation cycle (Figure 11). The first and most sensitive state occurs early in translocation and since FA does not inhibit GTP hydrolysis by EF-G (105), we tentatively identified this state as a pre-translocation complex after GTP hydrolysis but before mRNA movement. From this state FA-free as well as FA-bound ribosomes rapidly proceeded to a downstream state from which FA was slowly released and to which it could rebind with about thir-tyfold lower efficiency than to the first state. The average time of release of FA from this downstream state, which we tentatively identified as the inter-mediate translocation complex visualized by cryo-EM (63), was about 9 seconds. The average release time of FA from the post-translocation state, as visualized by X-ray crystallography (65), was shorter, only about 6 seconds,

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and we therefore concluded that there were in total three FA-sensitive states. The EF-G- and FA-bound post-translocation complex can be reached either by binding of FA to the EF-G-bound post-translocation complex after com-pletion of mRNA and tRNA movement, or by rebinding of EF-G and FA to the factor-free post-translocation ribosome. In the latter case EF-G and FA act as competitive inhibitors of ternary complex binding. This competitive inhibition was weak and not likely to play an important role in vivo, except when ternary complex concentrations are low. The higher FA binding effi-ciency to the first sensitive state compared to the downstream states may result from a longer life-time of the first state, but may also be due to a larger drug binding rate.

Figure 11. The complete mechanism of translocation inhibition by FA. FA has three modes of action, the first of which (I) is the efficient targeting of an early transloca-tion state, presumably the state after GTP hydrolysis but before mRNA movement. From this state the ribosomes proceed to an intermediate translocation state from which FA is released and to which it can also rebind less efficiently, in its second mode of action (II). The third mode of action (III) is the binding of FA to the post-translocation complex. This is achieved either by binding of FA after completed translocation or by rebinding of EF-G and FA to the post-termination complex after EF-G release, in competition with ternary complex binding.

We found that the concentration of FA required to double the elongation cycle time was only about 0.6 µM. This is more than two-hundredfold lower than a previous estimate by Savelsbergh et al. (37). In paper II we were able to explain the large discrepancy between the two results, which originated from poor experimental design in the previous study (37). The authors per-formed multiple turnover translocation experiments, in which 0.02 µM EF-G was let to cycle on 0.2 µM pre-translocation complexes at varying FA con-centrations and translocation was detected by puromycin reactivity. This setup allowed only ten EF-G cycles, giving a minimal EF-G cycling time of 6 s for the reaction not to be completed within the 1 min incubation time. If the reaction was over before the end of incubation, this would itself mask the

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inhibition. We showed in paper II that the FA concentration required for doubling the elongation time (the apparent Ki-value, K50%) depends on the cycling time in the absence of FA, as K50% = τAG·qF2·KI1, where τAG is the EF-G cycling time in the absence of FA, qF2 (0.11 s-1) is the release rate of FA and KI1 (120 µM) is the inhibition constant for binding of FA to the first FA-sensitive state. Using these parameter estimates and an uninhibited EF-G cycling time of 6 s it is possible to calculate the lower detection limit for the K50%-value by the puromycin based FA inhibition assay as 80 µM. Further-more, by analyzing only a single time point the slow establishment of the steady state, characteristic of tight binding inhibitors (106), was not taken into account. This may lead to underestimation of the EF-G cycling time and further overestimation of the K50%-value. Based on their very high K50%-value for FA inhibition of translocation (200 µM) and their estimate of a KI-value of 0.1 µM for ribosome recycling the authors concluded that the main inhibitory effect exerted by FA in vivo must be on ribosome recycling. Our estimate of a much stronger elongation inhibition prompted reassessment of the impact of FA in ribosome recycling and of the in vivo drug effect.

In paper IV we determined the complete kinetic mechanism for FA inhibi-tion of ribosome recycling including all parameters related to FA binding and release (Figure 12). We found that FA binds to EF-G on the post-termination complex both in the presence and absence of RRF. Time traces of ribosome splitting in the presence of FA displayed similar biphasic behav-ior as observed for translocation. The same data analysis methodology was used and the average times of the fast (τA) and slow (τI) phases and the frac-tion of FA-bound ribosomes (pI) were estimated for each time trace. The average release time of FA was about 5 s, similar to the release times in translocation (6 or 9 s). However, in contrast to the translocation case the total time of ribosome recycling increased linearly with the FA concentra-tion, as predicted by the total time expression derived from our model (Figure 12). Stronger inhibition of the recycling reaction was observed at a high EF-G-to-RRF concentration ratio, as a result of increased binding of EF-G and FA to the RRF-free post-termination complex. This was due to a sharper increase in both the fraction of FA-bound ribosomes (pI) and in the average inhibition time (τI) with the FA concentration. The FA concentration required to double the ribosome recycling time (the Ki-value) varied with the EF-G and RRF concentrations, but had a minimum of 0.17 µM. Under in vivo relevant conditions we estimated a Ki-value of about 0.3 µM, close to the value obtained for translocation inhibition (0.6 µM). We found that the GTP consumption of the ribosome recycling reaction was independent of the FA concentration. This implied that the complex with RRF, EF-G and FA bound to the post-termination ribosome was stable until FA release. Prema-ture release of RRF from this complex would require release of EF-G and FA followed by rebinding of both RRF and EF-G for ribosome splitting to

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occur, resulting in an additional round of GTP hydrolysis by EF-G. Further-more it implied that splitting competence was not lost due to FA binding. If the complex would not be able to split after the release of FA, EF-G release followed by EF-G(GTP) binding to the RRF-bound post-termination com-plex and GTP hydrolysis would be required to achieve splitting.

Figure 12. The complete mechanism of FA inhibition during ribosome recycling. We extended our kinetic mechanism for ribosome recycling (Figure 10) to include the steps related to FA inhibition. FA binds to the EF-G(GDP)-bound post-termination complex with rate constant kFA1 and is then released slowly with rate constant, qFA1. FA also binds to the RRF- and EF-G(GDP)-bound post-termination complex with rate constant kFA2 and is slowly released with rate constant qFA2.

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In vitro experiments and the living cell

The overall aim of the work presented in this thesis has been to study fun-damental steps of bacterial protein synthesis in the test tube, but under condi-tions retaining full ribosome activity and in vivo like translation kinetics. This tradition dates back to the development of a buffer system optimized for efficient and accurate phenylalanine incorporation in polyU-directed protein synthesis (12). This “polymix” buffer system has to date been shown to support fast initiation (16), fast and accurate incorporation of amino acids (13), fast translocation (Paper I), fast termination (40) and fast and efficient splitting of the post-termination ribosome after completed synthesis and release of a protein (Paper III). In paper I we demonstrated translocation rates at saturating EF-G concentration of 22 - 35 s-1, fully compatible with an in vivo elongation rate of 15 - 20 amino acids per second (70-72). In paper III we measured ribosome recycling rates of up to 10 s-1 and predicted a maximally achievable ribosome splitting rate of 25 s-1, significantly higher than all previous estimates (37, 82).

In vitro studies of ribosome function are still often performed under condi-tions causing artificially slow reaction rates, incompatible with protein syn-thesis rates observed in vivo. The major explanations are low temperature, high magnesium ion concentrations and low concentrations of translation factors. High magnesium ion concentrations are detrimental for ribosome function and lead to, for example, slow translocation (Paper I) and inaccurate selection of aminoacyl-tRNAs in the ribosomal A site (13). Large differ-ences in reaction conditions between different studies make their results difficult to compare. Furthermore, perturbed ribosome function and artifi-cially slow reaction kinetics may give rise to unnatural side reactions, not relevant for the in vivo situation. We believe that our kinetic models of EF-G-dependent translocation (Paper I) and EF-G- and RRF-dependent recy-cling (Paper III) of bacterial ribosomes, as they exhibit in vivo compatible reaction rates, provide the best available basis for modelling of how these processes occur in the cell.

The intracellular ribosome concentration in E. coli is high, about 50 µM (1), and the EF-G concentration is around 80% of this (107-109). Our estimates of the Michaelis-Menten parameters (kcat = 22 s-1 and KM = 0.65 µM, Paper I) for translocation, together with the high rates of peptide bond formation

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observed previously (39), suggest that translocation is the slowest step of the elongation cycle. A major fraction of the elongation cycle time is spent in the EF-G-bound ribosomal states undergoing the large conformational changes required for movement of the mRNA and the tRNAs in relation to the ribosome. Thus, most of the EF-G present in the cell is likely bound to translating ribosomes at all times. Accordingly, using the concentrations and Michaelis-Menten parameters stated above and assuming an average peptide bond formation rate of 100 s-1, we estimated the intracellular free concentra-tion of EF-G to be only around 4 µM, yielding an overall elongation rate of 16 amino acids per second (Paper III).

Bacterial cells invest a large fraction of their resources in the synthesis of ribosomes and translation factors. The intracellular concentrations of the various translation factors are most likely optimized to achieve the highest possible rate and energy efficiency of protein synthesis to maximize the growth rate. There is a cost associated with increasing the concentration of any translation factor in the cell, which is compensated if it results in an increased rate of the catalyzed reaction leading to a large enough increase in the overall translation rate. When the rate increase becomes too small to compensate the cost, due to saturated factor binding or because another step dominates the overall translation time, the factor has reached its optimal concentration (110).

Apart from its role in elongation EF-G is also involved in the splitting of the post-termination complex together with RRF. As the synthesis of a protein requires several hundred translocation steps, but only a single recycling step, the EF-G concentration is likely tuned for fast translocation. The recycling reaction, together with the termination steps, only needs to be fast enough to clear the stop codon before the arrival of the next translating ribosome. The intracellular RRF concentration is probably set to achieve fast enough ribo-some recycling, but also to avoid futile idling and GTP hydrolysis by EF-G on the post-termination complex (Paper III). Interestingly, the in vivo con-centration of RRF is relatively high, about 15 µM (111, 112), although RRF catalyzes only a minor step in the synthesis of a complete protein. In paper III we showed that for each concentration of RRF there is an optimal EF-G concentration, which maximizes the recycling rate. At 15 µM RRF, the EF-G optimum is 4.5 µM, very close to the free EF-G concentration estimated from our translocation data. At these factor concentrations our recycling model predicts a recycling rate of about 5 s-1 and a GTP consumption of 4.6 GTP molecules per ribosome splitting event.

In papers II and IV we clarified the mechanisms for fusidic acid (FA) inhibi-tion of EF-G, in translocation and ribosome recycling. A previous study strongly stated that recycling inhibition must be the dominating effect exerted

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by FA in vivo (37). This conclusion was based on a very high inhibition con-stant for translocation (200 µM), i.e. the FA concentration required to decrease the translocation rate to half, and a very low inhibition constant for ribosome recycling (0.1 µM). In paper II we reassessed the translocation inhibition and found that the sensitivity of elongating ribosomes to FA, due to poor experi-mental design, had been overestimated several hundredfold. Under physiolog-ically relevant conditions we estimated an inhibition constant of 0.6 µM for translocation, and an inhibition constant of 0.3 µM for ribosome recycling (Paper IV). Although the recycling time increased more rapidly with the FA concentration it must be taken into account that the synthesis of an average size protein requires several hundred elongation cycles but a single recycling event. We showed that the recycling time will at physiologically relevant con-centrations of EF-G, RRF and FA always constitute a minor fraction of the total time required for the synthesis of an average protein.

We concluded that the major target for FA in vivo is ribosomes in the elon-gation phase. Due to the long residence time of FA only a small fraction of the ribosomes need to be inhibited to achieve a large increase in the overall elongation time. For example, at an FA concentration of 1.2 µM only 1% of the ribosomes were FA-bound but the average elongation cycle time in-creased as much as threefold. Although FA targets several states during translocation, the first state, tentatively identified as the pre-translocation ribosome after GTP hydrolysis by EF-G but before mRNA movement, is thirtyfold more sensitive than the downstream states. Targeting of this first state will thus at in vivo relevant concentrations of FA be the dominant effect of the drug. FA binding halts an elongating ribosome for on average 9 s, which is more than hundredfold longer than the time of an uninhibited elon-gation cycle. Such events are likely to induce queuing of trailing ribosomes on the mRNA, amplifying the inhibitory effect of the drug.

It has been found that overexpression of EF-G makes bacterial cells more sensitive to FA (113). Since the translocation inhibition by FA does not de-pend on the EF-G concentration this is peculiar. Two possible explanations arise from our work, firstly, EF-G and FA binding competes with and inhib-its the binding of ternary complexes to the post-translocation complex. This effect is normally very weak but may be significant at certain codons or un-der certain conditions when ternary complex concentrations are low. Sec-ondly, inhibition of ribosome recycling by binding of EF-G and FA to the post-termination complex becomes more prominent at high EF-G concentra-tions. A testable prediction from this latter hypothesis is that overexpression of RRF in the cell would relieve the increased FA sensitivity at high EF-G concentration.

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Future outlook

Our assay for determining the translocation time is unique in that it measures the average total time for all steps of EF-G action on the ribosome, from factor binding to factor release. Other methods have analyzed only the inter-vening substeps like GTP hydrolysis, Pi release, ribosome ratcheting and mRNA and tRNA movement. Due to large differences in reaction conditions (temperature, magnesium ion concentration, factor concentrations etc.) be-tween different studies the results are difficult to compare. Although chal-lenging, it would be interesting if multiple methods could be combined for analyzing various aspects of translocation in a single reaction system, prefer-ably a system with in vivo like function like ours. In this way the order and timing of events during translocation could hopefully be determined unam-biguously.

Due to its transient nature the RRF- and EF-G-bound post-termination com-plex has not been possible to visualize by conventional structure determina-tion methods, unless with heterologous factors (89). Fortunately, time-resolved cryo-EM methods, in which a reaction mixture can be applied to an EM grid and frozen within tens of milliseconds, are currently under devel-opment (114, 115). To determine the structure of the RRF- and EF-G-bound post-termination complex, and to follow the compositional and conforma-tional dynamics that lead to ribosome recycling, by such methods, we have started a project in collaboration with the group of Professor Joachim Frank at Columbia University. In paper III we determined the complete kinetic mechanism for ribosome recycling at 37 °C. In preparation for the time re-solved cryo-EM experiments, which are performed at room temperature, we repeated all experiments at 20 °C. Based on these results we performed computer simulations of the recycling reaction to optimize the experimental setup for maximizing the yield of the RRF- and EF-G-bound post-termination complex. Initial results from a time-resolved cryo-EM experi-ment with 140 ms incubation time shows that we were indeed, for the first time, able detect the RRF-and EF-G-bound post-termination complex with homologous factors by structure determination methods. At this incubation time about 40% of the ribosomes had been split, and as expected we found about equal amounts of the 30S and 50S subunits in the sample. Interesting-ly, the 30S subunit was after splitting found to be bound by either a tRNA or by IF3, strengthening the evidence for the ribosome recycling model, sug-

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gested in previous work from the Ehrenberg group, in which IF3 induces dissociation of the tRNA from the 30S subunit after separation of the ribo-somal subunits (33). More time points will be taken to allow for a kinetic analysis of the compositional dynamics of the complexes in the sample that can be compared to the biochemical results and to follow the conformational dynamics of the factors involved.

Another project of interest would be to investigate also the translocation process by time-resolved cryo-EM. All existing structures of the EF-G-bound pre-translocation complex and of intermediate translocation complex-es were determined in the presence of antibiotics or with non-hydrolyzable GTP analogues. Direct visualization of the conformational changes in EF-G on the ribosome during real time translocation would remove any artifacts resulting from long time incubation of the sample and the presence of antibi-otics or GTP analogues that make the functional interpretation ambiguous.

In papers II and IV we deciphered the mechanisms of fusidic acid (FA) inhi-bition of translocation and recycling of the bacterial ribosome. FA is an ex-ample of a tight binding inhibitor, which, due to its long residence time, in-duces characteristic biphasic kinetics of the inhibited reaction with a slow phase increasing in amplitude and decreasing in rate with the inhibitor con-centration. We developed a methodology for analyzing this type of complex kinetic behavior using mean time methods. This methodology was used for studying FA-inhibition of both translocation and ribosome recycling and can also be used for studying other tight binding protein synthesis inhibitors, for example viomycin which is an already ongoing project (Holm et al., un-published). How mutations in EF-G or in the ribosome confer resistance to FA, by increasing the release rate of the drug or by reducing its binding effi-ciency, can easily be discerned by these methods. An interesting question is whether naturally occurring resistance mutations relieve the inhibition of translocation and ribosome recycling to the same extent. If mutations coun-teracting one type of inhibition are more prominent it may indicate that this is the main target of FA inhibition in vivo.

We showed in paper IV that although ribosomes in the elongation phase and in the recycling phase are targeted with similar efficiency by FA, inhibition of the former likely dominates the growth rate reducing effect inside the cell. As explained in the previous section, the translocation inhibition may be stronger in vivo, than our in vitro estimates, as stalling of ribosomes may result in ribosome queues on the mRNA. We are currently investigating these effects by computer modelling (Taberman et al., unpublished) and by ribosome profiling experiments, the latter in collaboration with the group of Professor Zoya Ignatova at Hamburg University.

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Sammanfattning på Svenska

Ribosomerna är de partiklar i cellen som tillverkar proteiner. I en snabbt växande bakteriecell finns tiotusentals ribosomer och proteinerna som de tillverkar utför många livsviktiga processer, till exempel att katalysera ke-miska reaktioner (enzymer), reglera genuttryck, transportera joner genom cellmembranet eller att känna av signaler från cellens omgivning (recepto-rer). Proteiner är långa kedjor av aminosyror som fogats samman enligt den genetiska information som lagras i cellernas DNA. Enzymer, s.k. RNA-polymeraser, kopierar (transkriberar) en DNA-gen i form av en enkel-strängad RNA-molekyl, budbärar RNA (på engelska messenger RNA, mRNA) som ribosomerna binder till och använder som mall för att koppla samman aminosyrorna i rätt ordning. Detta görs med hjälp av adaptormole-kyler, transport-RNA (tRNA), som översätter (translaterar) en sekvens av tre nukleotider i mRNA (ett kodon) till en viss aminosyra.

Proteinsyntesen har fyra faser, initiering, elongering, terminering och re-cycling där olika translationfaktorer (hjälparproteiner) är inblandade. Ri-bosomen består av två subenheter och har tre tRNA-bindningsplatser, ami-noacyl- (A), peptidyl- (P) och exit-siten (E). Under initieringen binder den lilla subenheten (30S), med hjälp av proteiner som kallas initieringsfaktorer, till ett mRNA. En viss sekvens i mRNA, den s.k. Shine-Dalgarnosekvensen, basparar med ribosomens eget RNA, rRNA, för att placera mRNAt rätt. Ett initierings-tRNA som bär den första aminosyran (formylmetionin, fMet) binder till startkodonet i P-siten. Sedan ansluter den stora subenheten (50S), initieringsfaktorerna dissocierar och ribosomen går in i elongeringsfasen. Ett ternärkomplex, bestående av ett tRNA, elongerings-faktor Tu (EF-Tu) och GTP, binder sedan till A-siten, EF-Tu hydrolyserar GTP och lämnar därefter ribosomen. Om ett felaktigt tRNA har bundit till kodonet i A-siten kommer det med hög sannolikhet dissociera, men om det är korrekt bildas snabbt en peptidbindning mellan dess aminosyra och peptiden (aminosyrakedjan) på tRNAt i P-siten. Peptiden, nu förlängd med en aminosyra, förs då över till tRNAt i A-siten och ribosomen går in i sitt roterade tillstånd, där de två subenheterna är vridna ca. 6° i förhållande till varandra. De två tRNA-molekylerna måste nu förflyttas från A- respektive P-siten till P- respektive E-siten för att en ny aminosyra ska kunna fogas till den växande peptidked-jan. Detta sk. translokeringssteg katalyseras av elongeringsfaktor G (EF-G) som strax efter att ha bundit till ribosomen hydrolyserar en GTP-molekyl.

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Energin som då frigörs omvandlas till konformationsförändringar i EF-G som leder till tillbakarotation av ribosomens subenheter, förflyttning av de två tRNA-molekylerna och förskjutning av mRNAts position på ribosomen med ett kodon. Cykler av peptidbindningsbildning och translokation uppre-pas till dess att ribosomen når ett stopkodon. I termineringsfasen binder en releasefaktor (Typ 1) till ribosomens A-site och hydrolyserar bindningen mellan peptiden och tRNAt i P-siten, så att det färdiga proteinet kan lämna ribosomen. Ytterligare en releasefaktor (Typ 2), binder därefter till riboso-men för att frigöra den första releasefaktorn. Posttermineringskomplexet som då återstår innehåller mRNA och ett deacylerat tRNA i P-siten, dvs. ett tRNA utan vare sig aminosyra eller peptid. Detta komplex splittras i re-cyclingfasen av EF-G och ribosome recycling factor (RRF) som separerar de två subenheterna från varandra för att frigöra dem för initiering av protein-syntes på ett nytt mRNA. Initieringsfaktor 3 (IF3) binder till 30S-subenheten och katalyserar tRNA- och mRNA-dissociation samt förhindrar subenheter-na från att återbinda till varandra i förtid.

I mitt avhandlingsarbete har jag studerat de två faser av proteinsyntesen där EF-G spelar en viktig roll, elongering och recycling. Alla experiment har utförts i provrör (in vitro), men under förhållanden som är optimerade för att ribosomerna ska behålla den funktion och aktivitet som de har inuti cellen (in vivo). I artikel I utvecklade vi en metod för att mäta genomsnittstiden för ett translokeringssteg vid valfri position längs mRNA. Till skillnad från tidi-gare metoder, som bara kunnat följa delsteg i translokeringsprocessen, mätte vi med vår metod den totala tiden för alla EF-G-beroende steg, faktorbind-ning, GTP-hydrolys, mRNA- och tRNA-förflyttning, subenhetsrotation samt EF-G-dissociation. Vi kunde visa att det första translokeringssteget efter initieringen var långsammare än ett translokeringssteg 15 kodoner längre ned längs mRNAt. Vid låg koncentration av magnesiumjoner, såsom i cellen, var translokeringen snabb, med hastigheter förenliga med de elongeringshastig-heter som uppmätts in vivo. Hög koncentration av magnesiumjoner är för-ödande för ribosomernas funktion och leder till mycket långsam transloke-ring. Vi undersökte också en hypotes från en tidigare studie, där ribosomer-nas fördelning på mRNA inuti cellen analyserats, som föreslog att Shine-Dalgarno-lika sekvenser i mRNA påverkar hur fort ribosomerna transloke-rar. En starkare växelverkan mellan mRNA och ribosom skulle då ge en långsammare translokering, men någon sådan effekt kunde inte påvisas med vår metod.

I artikel III studerade vi hur EF-G och RRF samarbetar för att separera ri-bosomens subenheter från varandra under recyclingfasen. Vi kartlade den fullständiga mekanismen för detta och bestämde hastighetskonstanterna för alla delsteg i processen. Vi kunde visa att RRF måste binda till ribosomen innan EF-G för att splittring skulle ske. EF-G tävlar med RRF om att binda

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till posttermineringskomplexet, vilket leder till långsam splittring samt onö-dig hydrolys av GTP vid höga EF-G koncentrationer. Den maximala splitt-ringshastigheten, som är 25 s-1, uppnås vid höga koncentrationer av både EF-G och RRF och när RRF-koncentrationen samtidigt är mycket högre än EF-G-koncentrationen. Antalet GTP-molekyler som konsumeras per splittrings-händelse minskar med ökad RRF-koncentration och når tillslut ett värde nära en GTP per splittring, vilket avspeglar hög sannolikhet för splittring av post-termineringskomplexet när både RRF och EF-G har bundit. Vår biokemiska kartläggning av recyclingmekanismen, i kombination med en nyutvecklad metod för tidsupplöst kryo-elektronmikroskopi, har möjliggjort strukturbe-stämning av det RRF- och EF-G-bundna posttermineringskomplex.

Fusidinsyra är ett antibiotikum som inhiberar proteinsyntesen genom att binda till ribosombundet EF-G under både translokeringen och i recyclingfa-sen. I artikel II undersökte vi hur fusidinsyra påverkar translokeringen och fann att tre olika fusidinsyrakänsliga tillstånd förekommer i varje elongeringscykel. Det första och känsligaste, som nås tidigt i translokering-en, identifierades preliminärt som det stadium som uppnås efter GTP-hydrolys men före mRNA-förflyttning. Från detta stadium fortsätter riboso-merna snabbt till ett tillstånd från vilket fusidinsyran kan frigöras, men till vilket den även kan återbinda. Strukturen av ett sådant intermediärt translo-keringstillstånd har tidigare bestämts med kryo-elektronmikroskopi. Det tredje fusidinsyrabundna tillståndet, vars struktur bestämts med röntgen-kristallografi, nås antingen genom fusidinsyrabindning efter slutförd tRNA- och mRNA-förflyttning eller, efter att EF-G har lämnat ribosomen, genom återbindning av EF-G och fusidinsyra till posttranslokeringskomplexet. I det senare fallet fungerar EF-G och fusidinsyra som kompetitiva inhibitorer av ternärkomplexbindning och påföljande peptidbindningsbildning. Denna ef-fekt visade sig dock vara svag, men kan möjligen bli viktig i vissa situationer när ternärkomplexkoncentrationerna är låga. Varje gång fusidinsyra binder till EF-G förblir den bunden i genomsnitt 5 till 10 sekunder. Eftersom denna tid är ungefär hundra gånger längre än genomsnittstiden för en oinhiberad elongeringscykel räcker det att en mycket liten andel av ribosomerna binder fusidinsyra för att uppnå en stark effekt. Vi fann att redan vid en antibiotika-koncentration av 0.6 µM ökade genomsnittstiden för en elongeringscykel till det dubbla trots att mindre än en procent av ribosomerna var bundna till fusidinsyra. Detta resultat stod i motsats till en tidigare studie som visat att det krävdes så mycket som 200 µM fusidinsyra för att dubblera tiden för en elongeringscykel. Samma studie innehöll även en uppskattning av fusidinsyrakänsligheten i recyclingfasen och visade att så lite som 0.1 µM fusidinsyra krävdes för att dubblera recyclingtiden. Baserat på dessa resultat drog författarna slutsatsen att den huvudsakliga effekten av fusidinsyra i cellen är kopplad till inhibering av recycling. Vi kunde dock visa hur under-målig experimentutformning i den tidigare studien lett till en underskattning

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av fusidinsyrakänsligheten i elongeringsfasen, vilket återigen gjorde fusidinsyrans huvudsakliga effekt i cellen till en öppen fråga.

I artikel IV kartlade vi den fullständiga mekanismen för fusidinsyrans inhi-berande effekt i recyclingfasen. Vi visade att fusidinsyra kan binda till EF-G både i närvaro och frånvaro av RRF på posttermineringskomplexet. I båda fallen var bindningstiden ca. 5 s, dvs ungefär lika lång som i elongeringsfal-let. Fusidinsyrans inhiberande effekt ökade med ökande koncentrationskvot mellan EF-G och RRF, på grund av ökad sannolikhet för bindning av EF-G och fusidinsyra till posttermineringskomplexet. Hur mycket fusidinsyra som krävdes för att dubbla recyclingtiden varierade alltså med koncentrationerna av de två faktorerna, men var cirka 0.3 µM vid förhållanden som liknar de i cellen. Våra resultat visar således att fusidinsyra inhiberar ribosomer i elongeringsfas och i recyclingfas ungefär lika effektivt. Dock måste man beakta att det för att tillverka ett genomsnittligt protein krävs flera hundra elongeringscykler men bara ett recyclingsteg. Trots att tiden för recycling ökar snabbare med fusidinsyrakoncentrationen än tiden för en elongeringscykel så kommer recyclingtiden, vid fysiologiskt relevanta kon-centrationer av fusidinsyra, aldrig att utgöra mer än en liten andel av den totala tiden för att tillverka ett protein. Den huvudsakliga effekten av fusidinsyra bör alltså vara inhibering av ribosomer i elongeringsfasen. Denna inhibering är sannolikt ännu starkare i cellen än den effekt vi uppskattat in vitro eftersom de långa pauser som fusidinsyran orsakar i ribosomernas rö-relse längs mRNAt kan leda till köbildning när bakomvarande ribosomer hinner ikapp den ribosom som stannat upp. Baserat på resultaten i artikel II och IV har vi nu påbörjat en studie för datormodellering av fusidinsyrans effekt på proteinsyntesen i cellen. Vi planerar också att utföra experiment som visar hur fördelningen av ribosomer på mRNA inuti cellen påverkas av fusidinsyra för att kunna visa vilket som är dess dominerande effekt och för att påvisa eventuella köbildningseffekter.

50

Acknowledgements

Thank you Måns! There have been ups and downs, but in the end I am very proud and happy about the things we achieved together. Thank you for shar-ing you unique ways of dealing with rate constants in particular and with life in general!

Suparna, thank you for scientific input and for always supporting me.

Thanks to 3H Biomedical for accepting me as an industry PhD student.

Thanks to Professors Joachim Frank and Zoya Ignatova for letting me work with your groups, although the fruits from that work are yet to be har-vested.

I want to thank all past and present members of the Molecular Biology program that I have worked with over the years, I will just mention a few. Thank you Misha for taking my results to another level, I wish I could un-derstand what you understand! Magnus for sharing your experimental knowledge, for comments on manuscripts and for always encouraging me. David for help with Matlab programming and data evaluation. Ikue and Vasili for pioneering the fusidic acid work. Ikue for tears and laughter and music, I really enjoyed having you as my friend and colleague! Kristin for common struggles in the early days, for arranging activities and fikas. Since you left, we miss you in the lab! Mikael for proofreading the thesis, for great parties, for “therapy” and discussions about anything between heaven and earth. Ray for supplying enormous amounts of components – I know, RRF was never your favorite! Harriet, Emmeli and Onur for present and future collaborations. Eugene and Erik for good work in the lab and for letting me practice my pedagogical skills on you. Thanks also to Akiko, for solving large and small problems!

I would also like to thank my family and friends, although in Swedish:

Tjejerna - Amina, Ullis, Katja, Christina och Malin, även om vi inte ses så ofta, uppskattar jag varje gång det blir av!

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Spelvänner och Dansvänner! För ”minisemestrar”, bortkoppling, hjärn-städning och ny energi!

Meta, för mysiga middagar och för att du håller ett öga på mig!

Per, du har varit ett stort stöd genom många år! Jag är stolt över alla bedrifter vi har klarat av tillsammans och glad över alla minnen från våra semesteräven-tyr!

Pappa, för att du finns där när det verkligen behövs och för allt du har lärt mig genom åren.

Mamma, jag saknar dig, det är så mycket jag hade velat dela med dig. Jag vet att du hade varit stolt över mig nu!

Magnus , du är den varmaste och mest omtänksamma person jag vet. Jag är så glad att jag får vara med dig.

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