Step 2. The 30S ribosomal subunit with IF3, or with all three bound initiation factors ("native" 30S particle), can associate with initiation region or ribosome-binding site (RBS) of mRNA. This region may be located near the 5'-end or far from it; in the case of polycistronic mRNA there may be several such regions along the mRNA chain. What is important is that this region is to be exposed to the interaction with the ribosomal particle and contain the polypurine Shine-Dalgarno sequence (SD), as well as the initiation triplet (AUG is the most preferable) at the proper distance downstream. In the absence of initiation factors, the 30S ribosomal subunit itself is also capable of recognizing the initiation region (RBS) of mRNA, but IF3 probably enhances the interaction:

30S:(IF3:IF1:IF2) + mRNA—► mRNA:30S:(IF3:IF1:IF2).

The mRNA bound in this complex is retained mainly by virtue of SD:ASD interaction and seems to be not properly established yet in the mRNA-binding cleft of the 30S subunit ("stand-by state").

Step 3. According to the above classical scenario, F-Met-tRNA is bound to the mRNA:30S complex at the next step. The binding is GTP-dependent. It is mediated by IF2. If IF2 is already sitting on the 30S particle, it can be activated by GTP and thus acquire the affinity for F-Met-tRNA, and then the F-Met-tRNA may join the complex:

mRNA:30S :IF3:IF1:IF2 + GTP + F-Met-tRNA-mRNA:30S :IF 1:IF2:GTP :F-Met-tRNA + IF3.

Codon-anticodon interaction of the initiation triplet of mRNA with the bound F-Met-tRNAfMet is believed to set the mRNA in the mRNA-binding site of the 30S subunit (Section 9.2).

If free IF2 with GTP encounters F-Met-tRNA in solution, they first form the ternary complex F-

ALEXANDER S. SPIRIN Met-tRNA:IF2:GTP, and then this complex binds to the mRNA:30S complex:

mRNA:30S:IF3:IF1 + F-Met-tRNA:IF2:GTP-30S:IF1:IF2:GTP:F-Met-tRNA + IF3.

There are indications that IF3 dissociates from the ribosomal particle upon F-Met-tRNA binding.

The alternative pathway has been also discussed when the "native" 30S particle with initiation factors first binds F-Met-tRNA, and then the complex formed interacts with the ribosome-binding region of mRNA:

30S:IF3:IF1:IF2 + F-Met-tRNA----*"-30S:IF1:IF2:GTP :F - Met- tRNA + IF3;

30S :IF 1:IF2:GTP:F-Met-tRNA + mRNA----• • mRNA:30S :IF 1:IF2:GTP :F-Met-tRNA.

In any case it is the anticodon of the bound F-Met-tRNA that searches for and finds the initiation codon downstream from the polypurine sequence and sets the ribosomal particle precisely at the start of the coding sequence.

Step 4. Now the initiating 30S complex is ready to join the 50S ribosomal subunit. IF1 seems to be released concurrently with the subunit association. The factor-binding site of the 50S subunit interacts with IF2 and induces the GTPase activity of the factor. GTP is hydrolyzed, resulting in the loss of the affinity of IF2 for F-Met-tRNA and the ribosome. Thus the 70S particle is formed precisely at the initiation codon of mRNA, with initiator F-Met-tRNA in the P site:

—»►•••[mRNA:30S:IF2:GTP:F-Met-tRNA:50S] + IF1----

—»►•••[mRNA:30S:IF2:GDP:F-Met-tRNA:50S] + IF1 + P{----

-• • mRNA:70S :F-Met-tRNA + IF1 + IF2 + GDP + P^

Step 5. The initiating 70S complex formed in the above reaction has the vacant A site, with the codon set there which immediately subsequent to the initiation codon. The P site is occupied with an analog of peptidyl-tRNA. Thus the 70S complex is competent to accept the first elongator aminoacyl-tRNA at its A site and perform the formation of the first peptide bond between the two substrates, initiator F-Met-tRNA and elongator aminoacyl-tRNA:

-• • mRNA:70S :F-Met-tRNA,Aa-tRNA :EF-Tu:GTP----

-»►•••mRNA:70S:F-Met-tRNAfAa-tRNAe + EF-Tu:GDP + Pi----

-►•• mRNA:70S :F-Met-Aa-tRNA + tRNAfMet + EF-Tu:GDP + P, e f i

This is the end of initiation and the beginning of elongation.

Fig. 15.6 presents schematically a tentative sequence of events during prokaryotic initiation of translation.






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