MECHANISM OF TRANSLATION

MECHANISM OF TRANSLATION

The synthesis of protein from mRNA involves translation of the language of nucleic acids into language of proteins. For initiation and elongation of a polypeptide, the formation of aminoacyl transfer RNAs is a prerequisite,

Formation of Aminoacyl rRNA

a)      Activation of amino acid

This reaction is brought about by the binding about by the binding of an amino acid with ATP and is mediated by specific activating enzymes known as amino acyl tRNA syntehtases or aaRs. As a result of this reaction between amino acid and adenosine triphosphate, mediated by specific enzyme, a complex (amino acyl-AMP- enzyme complex) is formed. Amino acyl-RNA synthetases are specific with respect to amino acids. For different amino acids, different enzymes would be required.

Aa_1­ + ATP             ^(Enzl)    (aa_1-AMP) Enz₁ + PP

b)     The transfer of amino acid to rRNA

The amino acyl-AMP-enzyme complex, formed during the step outlined above, reacts with a particular tRNA and transfers the amino acid to the tRNA. A particular amino acid would require a particular enzyme and a particular species of tRNA. This would mean that for 20 amino acids, at least 20 different enzymes and also atleast 20 different t-RNA species would be required.

(aa_1-AMP) Enz₁ + t-RNA₁               aa₁- t-RNA₁ + AMP + Enz₁

Initiation of Polypeptide

The initiation of polypeptide chain is always brought about by the amino acid methionine, which is regularly coded by the condon AUG,

In eukaryotes, formylation of initiating methionine is not brought about due to the absence of tRNA_f^met in plants and animals. Initiation in higher organisms will therefore, take place without formylation.

Initiation in eukaryotes

Initiation of polypeptide chain in eukaryotes is similar to that is prokaryotes, except the following minor differences. (i) In eukaryotes there are more initiation factors. They are named by putting a prefix ‘e’ to signify their eukaryotic origin. These factors are eIF1, eIF2, eIF3, eIF4A, eIF4B, eIF4C, eIF4D, eIF4F, eIF5 and eIF6. (ii) In eukaryotes, formylation of methionine does not take place. (iii) In eukaryotes, smaller subunit associates with initiator tRNA_i^met, without the help of mRNA, while in prokaryotes, generally the 30S-mRNA complex is first formed which then associates with f-met-tRNA_f^met.

Kozak’s ribosome scanning hypothesis for translation in eukaryotes

In 1983, Marilyn Kozak proposed a hypothesis for initiation of translation by eukaryotic ribosome. According to this hypothesis, 40S smaller subunit of a eukaryotic ribosome with its associated met-tRNA moves down the mRNA from 5’ end, until it encounters the first AUG. At this point, the 60S subunits join and the translation begins. The 80S ribosome, after reaching termination, releases protein and dissociates in two subunits.

Elognation of Polypeptide

The following three steps are important in the elongation process.

Binding of AA-rRNA at site ‘A’of ribosome (classical vs hybrid state models for translation)

In earlier classical model, each ribosome had two cavities, in which tRNA could be inserted. These were ‘P’ site and ‘A’ site. However, later a third cavity was suggested. F-met-tRNA_f^mef comes on ‘E’ site, to make ‘A’ site available for the next amino acyl tRNA (AA-rRNA).

Various steps of protein synthesis: (A-B) Attachment of tRNA-fmet-mRNA and smaller unit of ribosome, (C) Union of subunits of ribosomes, (D) Union of second

Another site called ‘R’ sites located on smaller subunit of ribosome, was proposed ‘R’ site plays a role in the improvement of accuracy of translation. The aminoacyl rRNA first binds to R site involving codon-anticodon pairing.

Later aminoacyl rRNA is flipped to ‘A’ site using energy from GTP molecule. During this flipping, tRNA is held only by condon-anticodon pairing. After formation of 70S initiation complex, the next amino acyl tRNA enters ‘A’ site. Elongation factors EF-Tu and EF-Ts participate. The elongation factor EF-Tu first combines with GTP and changes to an active binary complex, which binds with aa-tRNA, to form a ternary complex.

           

EF-Tu-Ts+GTP               EF+Tu.GTP+EF-Ts

                          ^{Binary Complex}

EF-Tu.GTP+aa-tRNA              EF-Tu..GTP.aa-tRNA

                                                                    ^{(Ternary Complex)}

(Hybrid states Models), it has been shown that above ternary complex actually binds in an A/P hybrid state, the anticodon binding to the A-site of the 30S subunit and the CCA end binding to the P-site of the 50S subunit as well as to the 30S subunit. The ‘P’ site is already occupied by f-met. tRNA_f^met or by a peptidyl tRNA. Following the GTP hydrolysis, EF-Tu.GDP+P are released from the ternary complex, permitting movement of CCA end of aa-tRNA into A site of the large 50S subunit. EF-Ts now displaces GDP in the EF-Tu.GDP binary complex and associates with EF-Tu, so that GTP can again associate with EF-Tu to start another cycle for the binding of aa-tRNA.

Formation of peptide bond

This is a catalytic reaction during which a peptide bond is formed between the free carboxyl group of the peptidyl tRNA at the ‘P’ site and the free amino group present with amino acyl tRNA, which is available at the A site. The 50S rRNA have peptidyl transferase activity, sothat the ribosome is described as a ribozyme.

According to this displacement model, peotidyl chain remains in a constant position relative to ribosome, while the tRNA moves during the peptide reaction. After the formation of peptide bond, the tRNA at ‘P’ site is deacylated and the tRNA at ‘A’ site now carries the polypeptide.

Translocation of peptidyl tRNA

From ‘A’ to ‘P’ site.

The peptidyl tRNA present at ‘A’ site is now Translocated to ‘P’ site. For translocation of peptidyl tRNA from ‘A’ site to P site, there are two models available: (i) According to two sites model, deacylated tRNA is liberated from ‘P’ site, and with the help of one GTP molecule and an elongation factor EF-G, the peptidyl tRNA is translocated from ‘A’ to ’P’ site. Thus according to this model, tRNA is either entirely in the A site or entirely in the P Site.

Various stages of protein synthesis: (E Formation of peptide bond and (F) Treansference peptidyle tRNA from A-site to P-site , (G) Attachment of third amino acid (serine) at A- site (H) Union of RF factor at A-site after the  completion of formation of peoptide bonds in between amino acids.

The requirement of EF-G and GTP for translocation was revealed by the use of antibiotic. The elongation factor EF-G binds to ribosome and is released on hydrolysis of GTP, which is a ribosomal function. EF-G and EF-Tu cannot bind to ribosome simultaneously, so that the entry of a fresh aa-tRNA on ‘A’ site and the translocation of peptidyl tRNA from ‘A’ to ‘P’ site has to follow each other and cannot occur simultaneously.

In eukaryotes, the elongation factor needed for translocation is called eEF-2, for the formation of one peptide bond. One ATP molecule and two GTP molecules (one for transfer of aa-tRNA to ‘A’  site and the other for translocation of peptidyl tRNA from ‘A’ to ‘P’ site) are required.

Termination of Polypeptide

Terminations in mRNA with stop condon

Termination of the polypeptide chain is brought about by the presence of any one of the three combination condons, namely UAA,UAG and UGA. These termination condons are recognized by one of the two release factors RF1 and RF2. The release factors to act on ‘A’ site, since suppressor rRNA capable of recognizing by entry at ‘A’ site. A third release factor RF3 stimulate the action of RF1 and RF2 in a GTP-dependent and condon independent manner GTP molecule is hydrolysed during release of a polypeptide, when RF3 stimulates RF1 amd RF2. For release reaction, the polypeptidyl tRNA must be present on ‘P’ site and the release factors help in splitting of the carboxyl group between the polypeptide and the last tRNA carrying this chain. Polypeptide is thus released and the ribosome dissociates into two subunits with the help of ribosome release factor or RRF.

It has been shown that the translation apparatus in chloroplasts and mitochondria differs from that in cytoplasm in eukaryotes in the following respects. (i) Ribosomes in these organelles are smaller in size than these in cytoplasm. (ii) The tRNAs are specific and differ, the number of tRNAs in mitochondria being 22 as against 55 in cytoplasm. (iii) Initiation of translation takes place by formyl-methionyl tRNA both in chloroplasts and mitochondria, although no formylation takes place in cytoplasm. (iv) Translation in chloroplasts and mitochondria can be inhibited by chloramphenicol.

Modification of Folding of Released Polypeptide

Modification of released polypeptide

After translation, the released polypeptide is modified in various ways.

Due to the action of certain other enzymes, exo-amino-peptidases, amino acids may be removed from either the N-terminal end or the C-terminal end or both.

The polypeptide chain singly or in association with other chains also folds into a tertiary structure. This problem of protein folding is sometimes described as ‘Second Half of the Genetic Code’.