Initiation in prokaryotes

Content
Expression of Gene : Protein Synthesis 4. Translation in Prokaryotes and Eukaryotes
Formation of amino-acyl tRNA
Initiation of polypeptide 
Initiation in prokaryotes
Initiation in eukaryotes
Kozak's scanning hypothesis
Elongation of polypeptide
Binding of AA-tRNA at site 'A' of ribosome
Formation of peptide bond
Translocation of peptidyl tRNA from 'A' to 'P' site
Termination of polypeptide
Modification, folding and transport of released polypeptide
Translation in chloroplasts and mitochondria


Initiation in prokaryotes
The initiating codon always functions in combination with a Shine-Delgarno sequence (SD region of mRNA), which has homology with the 3' end of 16S rRNA (the ASD region) found in 30S subunit of ribosome. SD region is a part of ribosome binding site (RBS), which itself is a part of a longer region called translation initiation region or TIR (Fig. 34.3). The TIR helps in binding with ribosome, and also accepts formylated methionyl tRNA at the initiating codon. The initiation involves the following steps in E. coli.

Formation of formyl-methionyl-tRNAfmet (f-met-tRNAfmet) The binding of methionine with tRNA^"e/ takes place in the manner outlined above for other amino acids. Later, met-tRNAfmet undergoes formylation with the help of transformylase enzyme in the presence of a formate source formyl-tetrahydrofolic acid.
 
The prokaryotic translational initiation region (TIR), and ribosome binding site (RBS); the Shine-Delgarno region relative to start codon (4-12 bases away) is also shown, which has homology to 3' end of 16S rRNA (ASD region).
Fig. 34.3. The prokaryotic translational initiation region (TIR), and ribosome binding site (RBS); the Shine-Delgarno region relative to start codon (4-12 bases away) is also shown, which has homology to 3' end of 16S rRNA (ASD region).


Generation of 30S subunit and binding of IF3. Ribosomes engaged in protein synthesis are released from mRNA as 70S (or 80S) particles. These particles are dissociated into two subunits (30S, 50S) with the help of the protein called initiation factor 3 (IF3). IF3 then binds to 30S subunit to stabilize it, to be ready to bind to mRNA (Figures 34.1A and 34.4).


Formation of 70S initiation complex. Several components, which participate in the formation of 70S initiation complex (70S ribosome + mRNA + f-met-tRNAfmet) include the following : (i) 30S and 50S ribosomal subunits, (ii) the initiator tRNA (f-met-tRNAfmet), (iii) the TIR of the mRNA, (iv) the three initiation factors (IF1, IF2, IF3) and (v) a GTP molecule. The probable events during initiation are depicted in Figure 34.4. The 30S subunit of ribosome is complexed with one molecule each of the three initiation factors and then binds, in random order, the RNA and f-met-tRNAfmet. (Properties and functions of three initiation factors in E. coli are given in Table 34.2). This leads to the formation of a pre-ternary complex, which undergoes rearrangement to form the bona fide 30S initiation complex. This complex can either dissociate into its original components or will associate with 50S subunit to form a 70S initiation complex. The 30S-50S association is rendered irreversible by the initial ejection of IF1 and IF3 and subsequent release of IF2 after hydrolysis of a GTP molecule. After 30S-50S association, f-met-tRNAfmet gets located on the ribosomal P site (P = peptidyl site), so that it can form a peptide bond with the incoming aminoacyl tRNA encoded by the second mRNA codon on the ribosomal A site (A = aminoacyl site). The formation of the first peptide bond marks the transition to the elongation stage of translation.

Use of initiation factors IF1, IF2 and IF3 in the formation of initiation complex with 30S subunit and the release of these factors on union with bigger 50S subunit in bacterial systems; A, B, A and B' are in rapid equilibrium; step C is the most important rate controlling step.
Fig. 34.4. Use of initiation factors IF1, IF2 and IF3 in the formation of initiation complex with 30S subunit and the release of these factors on union with bigger 50S subunit in bacterial systems; A, B, A and B' are in rapid equilibrium; step C is the most important rate controlling step.

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