Protein Synthesis I and II

Jörg Bungert

352-273-8098

jbungert@ufl.edu

Objectives• Know the structure of mRNA, tRNA, and main characteristics of

ribosomes.

• Know and understand the steps involved in translation

initiation, elongation, and termination.

• Know ansd understand how translation is regulated globally or

in an mRNA specific manner.

Reading: Lodish 6th edition,

Protein Synthesis

The polymerization of amino acids for protein synthesis is carried out by the ribosome, a large

4 MD ribonucleoprotein complex. The formation of the peptide bond in the peptidyl

transferase center of the large ribosomal subunit is catalyzed by rRNA. In this regard, the

ribosome can be viewed as a ribozyme. However, many proteins are required and serve

structural or regulatory functions.

The four main steps in translation are:

1) Initiation

2) Elongation

3) Termination

4) Recycling

Many of the fundamental steps in translation are conserved between eukaryotes and

prokaryotes. This is particularly true for the elongation step of translation. The process of

initiation, however, varies significantly and is the main step at which regulatory mechanisms

control the global rate of translation or the translation of specific messages in the cell.

Although the ribosome is regarded as the protein synthesis machine, it requires translation

factors to engage with the mRNA, to select activated building blocks for polypeptide

synthesis, to terminate translation, and to recycle ribosomal subunits to new

templates.

Structure of Ribosome Subunits

Prokaryotes:70S Ribosome; large 50S SU contains 23S rRNA, 5S rRNA, and about 34 different proteins;

small 30SU, 16S rRNA and 21 different proteins. MW= 2,500,000

Eukaryotes: 80S, large 60 SU contains 28S rRNA, 5.8S rRNA, 5S rRNA, and 49 proteins; small 40S

SU contains 18S rRNA and 33 proteins. MW= 4,200,000

About two thirds of the mass is RNA and one third is protein. The structure of the RNAs determine the

overall shape of the ribosome.

Structure of the tRNA

Structure of glutaminyl-tRNA synthetase with its

tRNA. The enzyme firmly grips the anticodon,

spreading the three bases widely apart for better

recognition. At the other end, the enzyme unpairs

one base at the beginning of the chain, seen

curving upward here, and kinks the long acceptor

end of the chain into a tight hairpin, seen here

curving downward. This places the 2' hydroxyl on

the last nucleotide in the active site, where ATP

and the amino acid (not present in this structure)

are bound. The amino acid is first linked to AMP

forming the adenylated amino acid, which then is

transferred to a hydroxyl group on the sugar at the

3’end of the tRNA (activated ester linkage)

3’ 5’

3’5’

mRNA

anticodon

tRNA

codon

wobble

position

tRNA structure,

anticodon – green,

acceptor end - red

Wobble base pairing

between codon and

anticodon.

In eukaryotes

wobble codon base/

anticodon base:

U/G or I

C/G or I

A/U

G/C

Aminoacyl-tRNA synthetases, tRNA, and Wobble base-pairing

The amino acid is first linked to AMP forming the adenylylated amino acid, which is then transferred

to a hydroxyl group on the sugar at the 3’end of the tRNA (activated ester linkage).

Linking of Amino Acids to Specific tRNAs by Aminoacyl-tRNA Synthetase

Structure of the messenger RNA

There are many structural features of the mRNA that play important roles in the process of

translation, especially in the steps of initiation and perhaps recycling. Unlike prokaryotes, the

eukaryotic mRNAs lack the Shine-Dalgarno sequence. Instead, the small ribosomal SU interacts

with the 5’end and according to current models scans along the RNA until the initiation codon is

found in a specific sequence context, referred to as the Kozak sequence. The main structural

features required for or regulating the efficiency of translation initiation are the 5’cap and the poly

(A) tail. These are bound by translation factors that recruit the 40S SU to the 5’end of the mRNA.

Other sequences, often located in the 5’ or 3’ UTRs (green ovals), regulate the efficiency of

translation. Secondary structures, like hairpins, near the cap-site can reduce translation

efficiency and special translation factors with RNA unwinding activities are required to resolve

these structures. IRES, internal ribosome entry sites, are used to recruit the “preinitiation

complex” to the mRNA in a cap-independent manner. These sequences are not well defined and

range in length from 7 nucleotides to more than 100. Secondary structure may play a more

prominent role than primary sequence and may be recognized by special proteins that mediate

cap-independent translation initiation. Some mRNAs contain upstream open reading frames

(uORF), which often inhibit translation initiation.

The 5’cap and interacting proteins

The 5’ cap is recognized by the initiation factor complex eIF4F. This multimeric complex consists

of eIF4E, the cap binding protein; eIF4G, a large protein which interacts with eIF4E, proteins

bound to the poly (A) tail, and other translation initiation factors (considered a scaffold protein),

and eIF4A, containing ATPase and RNA helicase activity. b, c: cap/eIF4E/eIf4G ternary complex,

eIF4E is yellow, eIF4G fragment is blue; short helix in lower right corner (eIF4E), and structures in

eIF4G shown here are induced by the interaction of the two proteins. The m7GDP is stacked in

between tow tryptophan residues. The methyl group is recognized and interacts with eIF4E, the

affinity for GDB/GTP is much reduced compared to methylated GDP.

Poly (A) and interacting proteins

The mRNA precursor is processed at the 3’ end by specific endonucleolytic cleavage immediately

after transcription. The 5’ fragment will become the mRNA, the 3’ fragment is degraded. This

cleavage is mediated by the Cleavage and Polyadenylation Specificity Factor (CPSF), which

recognizes the polyadenylation signal AAUAAA. CPSF stimulates poly (A) polymerase to add As

to the end. PABPN1 (nuclear poly (A) binding protein) also assists in tethering the polymerase to

the RNA. In the presence of CPSF and PABPN, the polymerase can add up to 250 As to the end of

the message.

PABPN is removed from the mRNA in the nucleus and replaced by the cytoplasmic PABPC upon

which the mRNA is exported to the cytoplasm. PABPC is 70 kDa and contains 4 RNA recognition

motifs, the first two of which are important and sufficient for RNA binding. 12 nucleotides are

required for high affinity binding, but it covers about 25 nucleotides.

Cap, poly (A), and pseudo-circularization of mRNA

The interaction between PABPC and eIF4G leads to the circularization of the mRNA.

Although the cap is sufficient to recruit translation factors and the 40S ribosomal SU, the

presence of the poly (A) tail stimulates translation initiation significantly. The requirement

for both cap and poly (A) tail ensures that only intact RNAs are translated. In addition,

circularization may facilitate translation re-initiation. The interactions between the cap- and

poly(A) complexes mutually stabilize each other. PABPC also interacts with the ribosome

release factor eRF3, a GTPase involved in translation termination and polypeptide release. A

current model proposes that the simultaneous interaction of PABPC with eIF4G and eRF3

results in close neighborhood of termination codon and the cap-complex, with the 3’UTR

looped out.

Translation Initiation and Initiation Factors (eIFs)

Initiation can be divided into four

major steps:

1) Formation of a 43S

preinitiation complex

consisting of the 40S SU,

initiation factors, and Met-

tRNAi.

2) Recruitment of the 43S

complex to the capped 5’end

of the mRNA.

3) Scanning of the 5’ UTR and

start codon recognition.

4) Joining of the large 60S SU to

assemble the translation

competent

80S ribosome.

Translation Elongation and Termination

Translation elongation is extremely conserved between prokaryotes, eukaryotes, and archae

bacteria. Aminoacyl t-RNAs are carried to the A (Acceptor) site as part of a ternary complex with

eEF1A and GTP. Base pairing between codon and anticodon, conformational changes, and GTP

hydrolysis ensure that only correct tRNAs are selected. Base pairing induces three bases from

the rRNA of the small SU to swing out and contact resulting mRNA/tRNA duplex, this activates the

GTPase activity. The peptidyl transferase center (P-site) catalysis formation of the peptide bond.

The following translocation is mediated by eEF2 and GTP hydrolysis. eEF1B is a multi factor

complex, which exchanges the GTP in eEF1A. No exchange factor has been described for eEF2.

eEF3 is unique to fungi and facilitates the release of the de-acylated tRNA from the E-site (Exit

site) and also stimulates the binding of eEF1A and tRNA to the A-site. The release from the E-site

requires ATP hydrolysis. Although, a similar factor has not been found in higher eukaryotes, the

exit from the E-site also requires ATP hydrolysis in these organism suggesting the presence of a

comparable activity to eEF3.

Translation termination is the consequence of stop codon recognition, for which no tRNA is

available. Instead, a class I release factor, eRF1, binds/decodes any of the three stop codons

(UAA, UAG, UGA). The recognition of the stop codon by eRF1 also involves the +4 position. Class

II release factor eRF3, a GTPase, stimulates the function of eRF1 as well as the hydrolysis of the

ester bond linking the polypeptide chain to the P-site tRNA. The hydrolysis requires water

molecules to enter the peptidyl transferase center, whereas elongating ribosomes must keep

water out during amid bond formation. eRF1 is thought to provide a channel for water molecules

to enter the transferase center.

Model of the prokaryotic ribosome

obtained by X-ray crystallography.

Shown are the small (30S) and large (50S)

subunits as well as the positions of the

acceptor (A), peptidyltransferase (P),

and exit sites. Protein components are

shown in darker color.

Structure of the Ribosome

Translation Elongation Continued

Translation Elongation

Translation Elongation Continued

Translation Elongation Continued

Translation Elongation Continued

Translation Elongation Continued

The Peptidyl Transferase Reaction

Peptidyl Transferase Center

(a) Large ribosomal subunit from

H. Marismortui with three intact

tRNAs in the E-, P, and A, site.

rRNA is white, ribosomal proteins

are yellow. (b) active site area

showing the peptidyl product (green)

bound to the A-loop (orange), and the

deacylated product (violet) in the

P-loop (dark blue). The N3 of A2486

is in close proximity to the 3’OH of

the CCA, and the base of U2620 has

moved close to the new peptide

bond and the 3’OH of A76.

Moore and Steitz, Annu. Rev. Biochem., 2003

Steps in the peptidyl transferase

reaction pathway. (a) a-amino group of

A-site substrate is positioned for pro-

R attack on carbonyl group of the ester

bond of the P-site substrate (green).

(b) Model of tetrahedral intermediate

with oxyanion pointing away from

A2486. (c) Structure of products of

peptidyl transferase reaction bound to

the peptidyl transferase center.

Translation Termination and Release Factors

Global Control of Translation

Global control of protein synthesis in response to various stress signals is generally achieved

by phosphorylation of translation initiation factors or proteins that regulate them. eIF2 consists

of the three subunits a, b, and g. The g-subunit binds GTP. A specific serine residue in the a-

subunit at position 51 is subject to phosphorylation. The phosphorylation of eF2 blocks the GTP

exchange reaction by reducing the dissociation rate of eIF2 from eIF2B, the GTP exchange

factor. Several stimuli lead to the phosphorylation of eIF2 including, haem depletion (by haem

regulated inhibitor), amino acid starvation (in yeast by GCN2), viral infection (by PKR, protein

kinase activated by double stranded RNA), and ER stress (by PERK).

The interaction of eIF4E with eIF4G is regulated by 4E-binding proteins (4E-BPs), which

competitively interact with eIF4E. Phosphorylation of 4E-BPs prevents association with eIF4E

and thus allows translation initiation. Extracellular signals, like insulin, triggers a cascade

leading to phosphorylation of 4E-BPs. The mammalian target of rapamycin (mTOR) is a critical

kinase that senses extracellular stimuly, amino acid availability, and the oxygen and energy

status to activate translation. It phosphorylates not only the 4E-BPs but also eIF4G and the S6

kinase, which phosphorylates eIF4B and enhances its interaction with eIF3. The MAPK pathway

also leads to phosphorylation of eIFs, e.g. eIF4B.

Global Control of Translation

RNA-dependent protein kinase PKR mediated phosphorylation of eIF2a

and shut-down of protein synthesis

Viral infection often leads to the accumulation

of double-stranded RNA byproducts. These

are recognized by PKR, which phosphorylates

eIF2a . Phosphorylated eIF2a forms high-

affinity sequestering complexes with its

nucleotide exchange factor eIF2B, which leads

to shut-down of protein synthesis. PKR

dimerization and autophosphorylation is

required for its catalytic activity.

A) PKR/eIF2a complex.

B) PKR dimer interphase

Viral pseudosubstrate inhibitors of PKR prevent shut-down

of protein synthesis

Most viruses have devised mechanisms to

prevent eIF2a phosphorylation by competitively

interacting with PKR. For example, Vaccinia

virus encodes the protein K3L, which mimics

the 3-dimensional structure of eIF2a and

competitively blocks eIF2a phosphorylation.

A. Model of K3L/PKR inhibitor complex based

on the binding mode of eIF2a. Regions of

structural similarities are colored grey.

B. Activation segment of PKR and interactions

with eIF2a.

mRNA Specific Regulation of Translation This type of regulation is carried out by proteins that bind specific sequences or structures in

mRNA, mostly in the 5’ or 3’UTR. These regulatory proteins generally repress translation. Iron

regulatory proteins (IRP) regulate translation of ferritin heavy- and light-chains, which encode

for the iron storage protein. In iron deficient cells, IRPs bind to the iron-responsive element (IRE), a

stem loop motif in the ferritin mRNAs, which blocks recruitment of the 43S complex (steric

hindrance). Other modes of regulation involve those that are mediated by proteins that interact with

the 3’ UTR and recruit proteins that interfere with eIF4 activity. These proteins can be regarded as

mRNA specific 4E-BPs as they all interact with eIF4E. Examples are Maskin, recruited by CPEB

(binds to uridine rich Sequence in maternal RNAs of oocyte), Cup, recruited by Bruno (binds to the

3’UTR of nanos in anterior parts of the developing drosophila embryo), and Bicoid, which directly

binds to an element in the Caudal 3’UTR in the anterior pole.

Sex-lethal (SXL) binds to poly (U) sequences in both 5’ and 3’ UTRs of msl-2 mRNA, which encodes

a component of the X-chromosome dosage compensation complex. SXL recruits a co repressor

(CR), which prevents the scanning of cap recruited 40S SU.

Translation of the ceruloplasmin or VEGF mRNAs is repressed by interferon-g. Repression is

mediated by the heteromeric GAIT (IFN-g activated inhibitor of translation) complex. GAIT is

composed of the 60S subunit protein L13a, glutamyl-, prolyl-tRNA synthetase, NS-1 associated

protein 1 (NSAP1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). IFN-g phosphorylates

L13a, which thendissociates from the ribosomal 60S subunit and forms the GAIT complex.

Phosphorylated L13a interacts with eIF4G and blocks its interaction with eIF3, thus inhibiting

recruitment of the 43S PIC.

mRNA Specific Regulation of Translation

Sonenberg, N, and Hinnebusch, A. G., Cell, 2009

Translation Control by Micro RNAs

Micro RNAs (miRNAs) are synthesized as primary transcripts in the nucleus and are processed

by Drosha, an RNase-III member. The 70 nucleotide pre-miRNA has a stem loop structure and is

exported to the cytoplasm. Here it is further processed by dicer into 22 nucleotide long miRNAs.

Dicer also generates siRNA, which degrade mRNAs due to perfect complementarity with the

target RNA and recruitment of the RISC complex (RNA induced silencing complex). A component

of the RISC complex is Argonaut, which is also part of the miRNA containing ribonucleoprotein

complex that repress translation. The main difference between miRNAs and siRNAs is the fact

that miRNAs are not perfectly complementary to the mRNA. If they are generated to be perfectly

complementary to the mRNA they behave like siRNAs and degrade the message. In contrast,

miRNAs bind to the 3’UTRs of target mRNAs and repress translation without degrading the RNA.

The mechanism by which translation is inhibited is unknown but must involve an elongation or

termination step as miRNAs were found to interact with polysomes. Initially miRNAs were

identified in Caenorhabditis elegans where they regulate expression of genes that are crucial for

developmental timing.

Interaction of signal recognition particle (SRP) with ribosome

The existence of signal sequence binding protein

for targeting to the endoplasmatic reticulum (ER) was

first proposed in the early 70’s. The binding

factor was subsequently identified as an 11S

ribonucleoprotein named Signal Recognition

Particle (SRP). The SRP has three activities. It binds

to the signal sequence of newly translated proteins,

it pauses translation elongation, and it promotes

translocation through the ER membrane by docking

to the SRP receptor at the ER. SRP has two domains.

The S domain consists of one half of the 7S RNA and

several proteins. SRP 54 protein is involved in

recognizing the signal sequence and interacts with

the SRP receptor in a GTP-dependent manner.

The signal sequence is recognized near the peptide

exit site of the large ribosomal SU. The Alu domain

consists of 5’ and 3’parts of the 7S RNA and several

proteins. It mediates elongation arrest to allow docking

of the ribosome to the SRP receptor.

Cryo-EM map of SRP bound to the RNC

(ribosome nascent chain complex). 40S SU

in yellow, 60S SU in blue, P-site tRNA in green,

SRP in red. C1-C6: assigned positions of RNC-SRP

Contacts; h1 and h2, hinges of the SRP 7S RNA; ST,

Stalk, SB, stalk base.Halic et al., Nature, 2004

Halic et al., Nature, 2004

Signal Sequence Dependent SRP Ribosome Interactions

SRP 54 has three domains, the N-terminus (N), the GTPase domain (G) and a methionine rich C-

terminal domain that interacts with the signal sequence. The binding induces a conformational change,

a kink in the 7S RNA possibly mediated by SARP 68/72, that allows interaction of the Alu domain with

the elongation factor binding site in the intersubunit space, where it causes arrest by competing with

elongation factors (EFS, elongation factor binding site).

Binding of Tetracycline to the 30S Prokaryotic Ribosomal SU

Several antibiotics, like tetracycline,

streptomycin, and hygromycin B all

target functionally important regions

in the 30S SU, mainly rRNA rich

regions associated with tRNA

interaction or movement through the

ribosome. Shown here is the

binding of tetracycline (Tet) to the

30S SU.

(a) Overview of primary and

secondary Tet binding sites (red).

The primary interaction of Tet is with

helix h34 of the 16S rRNA. The binding

site overlaps with the binding site

of the A-site tRNA and inhibits AA-tRNA

during the accommodation step, not the

initial binding of the ternary complex.

(b) Close-up view of the primary

binding site, with the position of the

A-site tRNA (red), mRNA (yellow),

and several helices of the rRNA.

(c) The secondary binding site does not

appear to play a role in Tet mediated

inhibition

Wilson and Nierhaus, 2003

Antibiotics targeting the large subunit

Crystal structures with antibiotics soaked into the crystal

of the large prokaryotic ribosomal SU have been solved with

14-membered macrolides (e.g. erythromycin) 16-membered

macrolides (e.g. carbomycin), 15-membered macrolides

(azithromycin) and nonmacrolide (e.g. blasticidin and

chloramphenicol) antibiotics. All of these antibiotics

bind in the proximal part of the polypeptide exit tunnel

adjacent to the PTC.

Moore and Steitz, 2003

Literature: Translation

1. Preiss, T., and M. W. Hentze (2003) Starting the protein synthesis

machine: eukaryotic translation initiation. BioEssays 25:1201-1211.

2. Von der Haar, T., Gross, J.D., Wagner, G., and J.E.G. McCarthy

(2004) The mRNA cap-binding protein eIF4E in post-transcriptional

gene expression. Nat. Struct. & Mol. Biol. 11:503-511.

3. Kuhn, U., and E. Wahle (2004) Structure and function of poly (A)

binding proteins. Biochem. Biophys. Acta 1678:67-84.

4. Gebauer, F., and M. W. Hentze (2004) Molecular Mechanisms of

translational control. Nat Review Mol. Cell Biol. 5:827-835.

5. Moore, P.B., and T. A. Steitz (2003) The structural basis of large ribosomal subunit

function. Ann. Rev. Biochem. 72:813-850.

6. Wilson, D. N., and K. H. Nierhaus (2003) The ribosome through the

looking glass. Ang. Chem. Intl. Ed. 42:3464-3486.

7. Halic, et al. (2004) Structure of the signal recognition particle interacting with the

elongation-arrested ribosome. Nature 427:808-814.

8. Hinnebusch, A.G. (2006) eIF3: a versatile scaffold for translation initiation

complexes. Trends in Biochm. Sci.

9. Dar, A.C., et al. (2005) Higher-order substrate recognition of eIF2a by the RNA-

dependent protein kinase PKR. Cell 122:887-900.

10. Chang, Y-F, J. S. Imam, and M. F. Wilkinson (2007) The nonsense-mediated decay

surveillance pathway. Ann. Rev. Biochem. 76:51-74.

11. Sonenberg, N., and A. G. Hinnebusch (2009) Regulation of Translation initiation in

Eukaryotes: Mechanisms and Biological Targets. Cell 136:731-745.