Protein Synthesis

Protein SynthesisSuvash C. Awal

The last step in the gene expression or the functional step of the genetic information.The only step of central dogma occurring exclusively in cytoplasm.

Requirement mRNAAminoacyl-tRNARibosomesOthers factors

RibosomesCell have tiny granular structures known as RibosomesRibosomes are Ribonucleo-Protein ParticlesRibosomes serves as workbenches, with mRNA acting as the blueprint in the process of protein synthesis.Ribosomes were first seen in cellular homogenates bydark-field microscopy in the late 1930s by Albert Claudewho referred to them as microsomes. It was not until themid-1950s, however, that George Palade observed them incells by electron microscopy, thereby disposing of the contention that they were merely artifacts of cell disruption.In 1955, Paul Zamecnik demonstrate it as the site of protein synthesis

The number of Ribosomes differs greatlyA rapidly growing E.coli cell may have as many as 15,000 to 20,000 ribosomes, about 15% of the cell massPolyribosome (or polysome): an mRNA bearing multiple ribosomes A single ribosome contacts with 30 nt, but mRNA can bind one ribosome for every 80 nucleotides.

Structure

Made up of RNA(rRNA) and proteins70S in prokaryotes and 80S in eukaryotesConsist of two subunitsLarge subunit 50S in prokaryotes60S in eukaryotesSmall subunit30S in prokaryotes40S in eukaryotes

Comparison of bacterial and eukaryotic ribosome

Secondary structure of rRNA

Svedberg Unit (S)The large and small subunit of ribosome are named according to the velocity of sedimentation when subjected to centrifugal force.The unit used to measure sedimentation velocity is Sevdberg (S).The larger value faster is the sedimentation velocity, hence larger the molecule.Named after inventor of ultracentrifuge Theodor Sevdberg.The sedimentation velocity is a function of a particles molecular weight, volume, size and shape

Functions Large subunitPeptidyl transferase center: that is responsible for the formation of peptide bondsSmall subunitDecoding center: where charged tRNA decode the codon units of the mRNA.Also the attachment of mRNA for the initiation before large subunit join.

Prokaryotic mRNAs have a ribosome binding site that recruits the translational machineryRBS (ribosome binding site): a short sequence upstream of the start codon that facilitates binding of a ribosome. AGGAGG. Also referred to as a Shine-Dalgarno sequence. Interacts with 16S rRNA (3 end containing anit Shine-Dalgarno sequence)In case that the start codon of downstream ORF overlaps the stop codon of upstream ORF, for example, with AUGA, translation of two ORFs is linked. This is known as translational coupling.

Some translational initiation sequences recognized by E. coli ribosomes

Eukaryotic mRNAs are modified at their 5 and 3 ends to facilitate translationThe 5 cap is a methylated guanine nucleotide at 5 end of mRNA. Recruits the ribosome.The ribosome moves in a 5 to 3 direction until it encounters an AUG in a process called scanning.The Kozak sequence (PuNNAUGG)(GCCRCCAUGG) interacts with initiator tRNA. Poly-A tail promotes efficient recycling of ribosomesEukaryotic initiation factor and poly-A binding protein recognise the 5 cap and recruit the 43S rRNA forming preinitiaiton complex.Cap-independent mode occur through the internal ribosome entry site (IRES) recruiting ITAFs (IRES trans acting factor)

Polio virus IRES mediated initiation

The large and Small subunits undergo association and dissociation during each cycle of TranslationThe ribosome cycle: the sequence of association and dissociation of the ribosome.

Peptide bonds are formed by transfer of the growing polypeptide chain from one tRNA to anotherThe ribosome catalyzes the formation of a peptide bond between the amino acids attached to tRNAs.Two consequences of the peptidyl transferase reaction: 1. The N-terminus of the protein is synthesized before the C-terminus. 2.The growing polypeptide chain is transferred from the peptidyl-tRNA to the aminoacyl-tRNA.No ATP is required, but ATP is spent during tRNA charging reaction.

Ribosomal RNAs are both structural and catalytic determinants of the ribosome Ribosomal RNAs are not simply structural components but directly responsible for catalytic activity.The peptidyl transferase center and the decoding center are composed almost entirely of RNA. Most ribosomal proteins are on the periphery of the ribosome.

The peptidyl transferase center

The decoding center

The ribosome has three binding sites for tRNAThe A site is for the aminoacylated-tRNA.The P site is for the peptidyl-tRNA.The E site is for the exiting tRNA.Each tRNA binding site is at the interface between the large and the small subunits of the ribosome.

Channels through the ribosome allow the mRNA and growing polypeptide to enter and/or exit the ribosomeThere are two narrow channels in the small subunit, one for entry and the other one for exit of mRNA. only wide enough for unpaired RNA to pass through.There is a kink in the mRNA between the two codons. The incoming aminoacyl tRNA cannot bind to bases immediately adjacent to the vacant A site codon.A channel in the large subunit provides an exit path for the newly synthesized polypeptide chain. The size of the channel limits the folding of the growing polypeptide chain. The polypeptide can form an alpha helix in the channel.

In the ribosome there are THREE STAGES and THREE operational SITES involved in the protein production line.The three STAGES are Initiation, Elongation andTermination.The three operational or binding SITES areA site for attachmentP site for peptide formationE site for exit (only on large subunit)

Table of binding sites, positions and functions in a ribosome

Initiation

In prokaryotesThe initiating 5 AUG recruit tRNAfMet that code for N-formylmethionine rather methionine in the internal codon sequence by tRNAMet.The addition of N-formyl group to the amino group of methionine by the transformylase prevent fMet from entering interior positions in a polypeptide.

Requirement 30S ribosomal subunitmRNAInitiating tRNAfMetInitiating factors (IF-1, IF-2, IF-3)GTP50S ribosomal subunitMg++

Its a three step processStep one30S ribosomal subunit binds to IF-1 and IF-3(it prevent combing 50S prematurely)

16S subunit recognize the Shine-Dalgarno sequence and this mRNA-rRNA interaction drive the positioning of intial 5 AUG to the P site as A site is blocked by IF-1.( the tRNAfMet only join to P site and after that every other incoming aminoacyl-tRNA join in A site)

Step twoThe complex consisting of the 30S ribosomal subunit, IF-3 and mRNA is joined by both GTP-bound IF-2 and the initiating fMet-tRNAfMet.The anticodon of this tRNA now pairs correctly with the mRNAs initiation codon.

Step threecomplex combines with the 50S ribosomal subunit; simultaneously, the GTP bound to IF-2 is hydrolyzed to GDP and Pi, which are released from the complex.

All three initiation factors depart from the ribosome at this point.

Completion of the steps in produces a functional 70S ribosome called the initiation complex, containing the mRNA and the initiating fMet-tRNAfMet.

Translation initiation factorsIF1 prevents tRNAs from binding to the portion of the small subunit that will become part of the A-site.

IF2 is a GTPase (a protein that binds and hydrolyzes GTP) that interacts with three key components of the initiation machinery: the small subunit, IF1, and the charged initiator tRNA (fMet-tRNAifMet). By interacting with these components, IF2 facilitates the association of fMet-tRNAifMet with the small subunit and prevents other charged tRNAs from associating with the small subunit.

IF3 binds to the small subunit and blocks it from reassociating with alarge subunit. Because initiation requires a free small subunit, the binding of IF3 is critical fora new cycle of translation. IF3 becomes associated with the small subunit at the end of a previous round of translation when it helps to dissociate the 70S ribosome into its large and small subunits.

Initiation in EukaryotesEukaryotic initiation process is far more complicated than prokaryotes, involving atleast 12 different initiation factors (designated eIFn; e for eukaryotic)

the small subunit is already associated with an initiator tRNAwhen it is recruited to the capped 50 end of the mRNA. It then scans along the mRNA in a 5-3 direction until it reaches the first 5-AUG-3 (kozak sequence).

binding of the initiator tRNA to the small subunit always precedes association with the mRNA

Initiation in Eukaryotes

43S preinitiation complex

Initiation factor required

Energy requiredFor prokaryotes1 ATP for aminoacyl-tRNA1 GTP for initiation

For eukaryotes3 ATP, one for aminoacyl-tRNA, one for RNA helicase, one for AUG scanning.2 GTP, one in release of initiation factor by eIF2, one by eLF5B for joining 60S larger subunit to 40S small subunit forming complete 80S complex.

Elongation

Elongation Ribosomes elongate polypeptide chains in a three-stage reaction cycle that adds amino acid residues to a growing polypeptides C-terminusDecoding, in which the ribosome selects and binds an aminoacyl-tRNA, whose anticodon is complementary to the mRNA codon in the A site. Transpeptidation, in which the peptidyl group on the P-site tRNA is transferred to the aminoacyl group in the A site through the formation of a peptide bond.Translocation, in which A-site and P-site tRNAs are respectively transferred to the P site and E site accompanied by their bound mRNA; that is, the mRNA, together with its base paired tRNAs, is ratcheted through the ribosome by one codon.

Elongation factor

Eukaryotic counterpartEF-Tu = eEF1AEF-Ts = eEF1BEF-G = eEF2

A Cycle of Peptide-Bond Formation Consumes Two Molecules of GTP and One Molecule of ATP One molecule of nucleoside triphosphate (ATP) is consumed by the aminoacyl-tRNA synthetase in creating the high-energy acyl bond that links the amino acid to the tRNA. The breakage of this high-energy bond drives the peptidyl transferase reaction that creates the peptide bond.

A second molecule of nucleoside triphosphate (GTP) is consumed in the delivery of a charged tRNA to the A-site of the ribosome by EF-Tu and in ensuring that correct codonanticodon recognition had taken place.

Finally, a third nucleoside triphosphate is consumed in the EF-G-mediated process of translocation.

Termination

Polypeptide formation end is signaled by the presence of one of three termination codons in mRNA (UAA, UAG, UGA).

Once any of these codon take the A site the termination process begins.

tRNA doesnt play role in it but the process start with the involvement of termination factors of release factors (RF)

When a mutation produces a termination codon in the interior of a gene, translation is prematurely halted and the incomplete polypeptide is usually inactive (nonsense mutations).

Gene can be restored to normal function if a second mutation either

Converts the misplaced termination codon to a sense codonSuppresses the mutation by the mutation in tRNA anticodon (nonsense supressors)

Release factor (RF)Stop codon recognition is depend upon the RF.Two class of RFClass I RF = RF-1, RF-2Class II RF = RF-3

Class I RF play role in recognition of stop codon and act as catalyst for the hydrolysis of peptidyl from peptidyl-tRNA

Class II RF are small G-protein and act by assisting class I RF in GTP-dependent manner.

Difference from elongationIn elongation codon recognition by aa-tRNA result in nucleophillic reaction in the peptidyl transferase centre (PTC), whereas in stop codon recognition by RF leads to hydrolysis of peptide-tRNA.

Elongation process have proof-reading mechanism to low frequency of error but RF in termination is independent of proof-reading.

Upon the stop-codon recognition the ester bond of peptidyl-tRNA is cleaved by hydrolysis in presence of water.

In prokaryotes, RF-1 recognize UAA, UAGRF-2 recognize UAA, UGA

The recognition depends upon the structure of RF-1, RF-2.RF is made up of 4 Domain.

Domain 1, bind to vicinity ribosomal GTPase associated centre

Domain II, role in stop codon recognition and also have PxT and SPF that differentiate for RF-1 and RF-2 respectively.

Domain III, spans between the functional centers of the small and large ribosomal subunit; the universally conserved GGQ motif implicated in the catalysis of peptidyl-tRNA in the peptidyltransferase center.

Domain IV, also role play in stop codon recognition.

Crystal structures of the 70S translation termination complexes bound with RF1 and RF2.

The codon reading head of the release factor comprises the N-terminal end of helix 5 and the conserved recognition loop formed between the -4 and -5 strands of the central -sheet of domain 2.

Three elements of the reading head are responsible for recognition of the three stop-codon nucleotides.

The N-terminal tip of helix 5 recognizes U via formation of specific H-bonds from the backbone of 5.Conserved amino acids of the recognition loop, including the PxT and SPF motifs of RF1 and RF2, respectively, define the specificity of release factors for the second nucleotide (A & G).

Interactions of the first two stop-codon nucleotides with release factors RF1 and RF2.

The N- and C-terminal ends of the recognition loop define the specificity for the third nucleotide located in the G530 pocket.

Recognition of the third stop-codon nucleotide by both RF1 and RF2 occurs separately from the first two nucleotides, in the G530 pocket of the decoding center.

Interactions of the third stop-codon nucleotide in the 70S translation terminationcomplexes bound with RF1 and RF2.

RF directly participate in catalysis of peptidyl-tRNA hydrolysisUpon recognition of a stop codon by RF, the ester bond bridging peptidyl and peptidyl-tRNA is hydorlyzed.

Domain-III tip with GGQ motif inserted into the PTC and contact the nucleotide of 23s rRNA, P-site, tRNA and it open a passage for a water molecule.

Process Recognition of stop codon by RF-1/RF-2.

Peptidyl hydrolysis form peptidyl-tRNA with introduction of water molecule.

Once the newly synthesized polypeptide has been released from the ribosome, the class II release factor RF-3, in its complex with GDP, binds to the ribosome. On binding to the ribosomeRF-1/2 complex, it exchanges its bound GDP for GTP. The resulting change in the conformation of RF-3, as seen in cryo-EM studies, causes it to bind more tightly to the ribosome and expel the RF-1/2

The interaction of RF-3GTP with the ribosome stimulates it to hydrolyze its bound GTP. The resulting RF-3GDP then dissociates from the ribosome. Subsequently, ribosomal recycling factor (RRF) binds in the ribosomal A site followed by EF-GGTP.

EF-G hydrolyzes its bound GTP, which causes RRF to be translocated to the P site and the tRNAs previously in the P and E sites to be released. Finally, the small and large ribosomal subunits separate, a process that is facilitated by the binding of IF-3, and RRF, EF-GGDP, and mRNA are released. The ribosomal subunits can then participate in a new round of initiation

Stop-codon recognition and peptidyl-tRNAhydrolysis are coordinated via conformational switch in release factorClass I release factors are high-fidelity enzymes.In order to achieve low error frequency (10-3-10-6), hydrolysis of peptidyl-tRNA has to be strictly coordinated with stop-codon recognition.This coordination is accomplished by preventing the docking of domain 3 into the PTC prior to recognition of a stop codon. a release factor initially interacts with the ribosome in a catalytically inactive conformation. Upon stop-codon recognition, a conformational change would occur resulting in the docking of the GGQ motif into the PTC

GTP Hydrolysis Speeds Up Ribosomal ProcessesWhat is the role of the GTP hydrolysis reactions mediated by the various ribosomally associated G proteins (IF-2, EF-Tu, EF-G, and RF-3 in bacteria)?

The high rate and irreversibility of the GTP hydrolysis reaction ensures that the various complex ribosomal processes to which it is coupled, initiation, elongation, and termination, will themselves be fast and irreversible. The ribosome utilizes the free energy of GTP hydrolysis to gain a more ordered (lower entropy) state rather than a higher energy state as often occurs in ATP-dependent processes

ReferenceVoet, D. & Voet, J. G. Biochemistry. Fourth Edition. 2011. John Wiley & Sons. INC.Watson, et. al. Molecular Biology of The Gene. Seventh Edition. 2014. Pearson Education, Inc. Korostelev, A. a. (2011). Structural aspects of translation termination on the ribosome. RNA (New York, N.Y.), 17(8), 14091421. Jackson, R. J., Hellen, C. U. T., & Pestova, T. V. (2010). The mechanism of eukaryotic translation initiation and principles of its regulation. Nature Reviews. Molecular Cell Biology, 11(2), 113127.