Translation is executed in six steps: (i) binding of mRNA to ribosome, (ii) aminoacylation, (iii) initiation, (iv) elongation, (v) termination and (vi) post-translational modification, (i) Binding of mRNA to ribosome
I. binding of mRNA to ribosome
Ribosomes occur in the cytoplasm in dissociated condition,ie, their smaller and larger subunits separated. In prokaryote, the transcription factor IF3 helps in dissociation of the two subunits of ribosome and then binds to the 30S subunit to prevent premature association of the two subunits. First, the mRNA binds to the smaller subunit. The smaller subunit has two binding sites: A site or aminoacyl site and P site or peptidyl site.
The incoming tRNA with its specific amino acid binds to the A site and the peptidyl tRNA carrying the elongating polypeptide binds to P site. The bacterial ribosome contains another site, the E site or Exit site to which the discharged tRNA or tRNA whose peptidyl has already been transferred binds before its release from ribosome.
The Prokaryotic mRNA has a leader sequence at its beginning just prior to the initiation codon AUG. This sequence is known as Shine-Delgarno Sequence (SD region), which has homology with the 3′-end of the 16S rRNA (ASD region) found in 30S subunit.
This complementarity ensures that the 30S subunit binds at the correct position of mRNA and the translation process starts from the beginning of mRNA. In eukaryote the 40S subunit enters at capped s’-end of the mRNA and then advances to the start codon by linear scanning.
Aminoacylation or activation of amino acid is the step in which all the twenty amino acids are linked to their specific tRNA in the cytoplasm. This reaction is catalyzed by the enzyme aminoacyl-tRNA sythctasc.
There are 20 different types of synthetases for 20 different amino acids. The amino acids recognized by two or more tRNA molecules are linked by the same enzyme. In the first step of aminoacylation, aminoacyl adenylate enzyme complex or aminoacyl- AMP-Enzyme is formed with the release of pyrophosphate.
AA, + ATP + Enzyme——————– AA-AMP-Enzyme + PP
Amino aminoacyl aminoacyl-adenylate
Acid synthetase – enzyme complex
In the second step this complex gets associated with the 3′-OII end (with unpaired CCA sequence) of specific tRNA molecule. The AMP is now hydrolyzed to form an ester bond between the amino acid and its specific tRNA, and the enzyme is also released.
The protein synthesis begins from the amino terminal end of the polypeptide, proceeds by the addition of amino acids through peptide bond formation and ends at the carboxyl terminal end. In prokaryote, the initiation amino acid is formylatcd mctheoninc while in cukaryotc it is mcthconinc. So in the prokaryote, there arc two types of tRNA for mctheoninc. One is tRNAP”ct for initiation carrying formyl metheonine and the other one is tRNAmet for carrying normal mctheoninc to growing polypcptide.
The initiation of polypeptide synthesis in prokaryote requires the following: l.raRNA, 2. 30S subunit of ribosome, 3. formylmetheonyl-tRNA (fmet-tRNAf”iet), 4. Initiation factors IF-i, IF-2 and IF-3, 5. GTP, 6. 50S ribosomal subunit and 7. Mg+2 the sequence of events that occurs during initiation are:
1. The smaller 30S subunit of ribosome binds to the transcription factor IF-3 that prevents premature association of the two ribosomal subunits.
2. The mRNA binds to 30S subunit through the interaction of SD region of mRNA and ASD region of ribosome so that the initiation codon AUG is correctly positioned at the P site of the ribosome.
3. The fMet-tRNAf*”1 (the specic tRNA aminoacylated to formyl methionine) binds to the AUG codon at the P site.The tRNAP”* is the only tRNA that binds to its codon present on the P site all other tRNA along with their respective amino acids bind to their codon present at the A site.
That is why AUG codons present as initiation codon codes for formylmethionine and when present at other position codes for normal methionine. It can be recalled that there are two types of tRNA molecules recognizing the same AUG codon but carrying two different forms of methionine.
4. The initiation factor IF-i Binds to the A site and prevents binding of any other aminoacyl tRNA to the codon at the A site during initiation.
5. Xow the GTP bound IF-2 (GTP-IF-2) and the initiating fMet-tRNAP1” join the complexof 3oSsubunit- IF3-IFi-mRNA.
6. Then 50S subunit joins the complex formed in the previous step. The GTP bound to IF-2 is hydrolysed to GDP and Pi. All the three initiation factors leave1 ribosome. This complex of 70S ribosome, mRNA and fMct-tRNAfMe: bound to initiation codon at P site is known as initiation complex.
Elongation step involves the addition of further amino acids so that the polypeptide chain would grow. This step requires the following: 1. the initiation complex, 2. different aminoacyl- tRNAs, 3 two elongation factors (EF-Tu and EF- Ts ) and 4.GTP. The elongation process takes place in three steps:
Step I- Binding of incoming aminoacyl
The incoming aminoacyl-tRNA binds to a complex of EF-Tu-GTP to result in a aminoacyl- tRNA-Tu-GTP complex. This complex binds to the A site of 70S initiation complex. Then GTP is hydrolysed to GDP and Pi and EF-Tu-GDP complex is released from the ribosome.The EF- Tu-GTP complex is regenerated and recycled by EF-Ts and GTP as follows:
EF-Tu-GDP + EF-Ts =EF-Tu -Ts +GDP EF-Tu-Ts + GTP = EF-Tu-GTP + EF-Ts Step II- Peptide bond formation This is a catalytic proccss during which a peptide bond is formed between two amino acids bound by their t RNA molecules to the A site and P site. This peptide bond is formed between the free carboxylic. Group of the X- formylmethionine group attached by its tRNA to the P site and the second amino acid bound by its tRNA to A site. The X- formyl group is transferred to the amino group of the second amino acid bound to its tRNA at A site. As a result, the tRNA
At the A site contains a dipeptide and that at the P site becomes empty. The enzyme responsible for the peptide bond formation is peptidyl transferase. In bacteria the 23S rRNA a component of the 50S ribosomal subunit is thought to carry out peptidyl transferase function.
Step III- Translocation
In this step the peptidyl tRNA bound to the A site comes to the P site of ribosome, the empty tRNA at P site comes to the E site and the A site is occupied by a new codon for the next incoming aminoacyl tRNA. This is accomplished by the movement of ribosome by one codon more in 5′ to 3′ direction of mRNA.
The translocation of ribosome requires EF-G (translocase) and GTP. The deacylated tRNA interacts with the E site mainly located on 50S subunit through its CCA sequence at the 3′-end. The two step transfer of tRNA molecules from A site to P site and from P site to E site could result from the reciprocating motions of the two subunits of ribosome.
This means that 50S and 30S subunits move alternately not simultaneously. Finally the deacylated (empty) tRNA is released to cytosol from the E site.
Termination of the synthesis of polypeptide is brought about by the presence of any one of the three termination codons on the mRNA. These termination codons are recognized by any one of the three release factors/ termination factors, RFi, RF2 and RF3. RFi and RF2 resemble the structure of tRNA and compete with it for binding to any one of the termination codon at the A site of ribosome.
This phenomenon is known as molecular mimicry.RFi recognises UAG and RF2, UGA. Both recognize UAA. When the A site of the ribosome encounters a termination codon occupied by a release factor instead of an aminoacyl tRNA, elongation of polypeptide stops. At the P site one tRNA with the polypeptide chain is bound to another codon.
The release factor RF3 coupled with GTP splits the peptidyl-tRNA bond. The polypeptide is thus released and the discharged or empty last tRNA is also released from the P site. The ribosome dissociates into 50S and 30S subunits. In eukaryote only one release factor cRFI is known.
The.fundamental mechanism of elongation and termination in eukaryotc resemble with that of prokaryotc.
(vi) Post-translational modifications
The released polypeptide is modified in various ways. The enzyme dcformylasc removes the formyl group from the first amino acid methionine. Then, due to action of exo-amino-peptidase enzyme certain amino acids may be removed from the X- terminal end or C-terminal end or both.
The polypeptide chains singly or in association with other polypeptides fold properly to take a tertiary structure to become functional proteins. Inhibition of transcription and translation
Transcription is selectively inhibited by several antibiotics and drugs. Actinomycin D inhibits the elongation process by intercalating between successive G-C base pairs. Translation is also selectively inhibited by several drugs and antibiotics in prokaryotes. However, these inhibitors are relatively harmless in eukaryotes. Puromycin C is one such important inhibitor which structurally resembles 3′ end of aminoacyl tRNA So this can participate in peptide bond formation producing peptidyl-puromycin. It dissociates from the ribosome and thus terminates translation.