ИСТИНА

Initiation of the protein biosynthesis in Eukaryotes is a complex multi-step process requiring concerted action of more than two dozens of components including two ribosomal subunits, Met-tRNAi, translation initiation factors and mRNA. Many aspects of this process are still unknown, and even the order of events during initiation step of translation is not absolutely clear. In most cases, the initiation starts with 40S ribosomal subunit, which binds the mRNA either near its 5’-end (in the case of the cap-dependent mRNAs) of at the specific internal structures (IRESes, in the case of the internal initiation) and begins to “scan” through the 5’-untranslated region in the 5’-3’ direction to locate an AUG codon. Once it reaches the AUG codon, it thought to stop irreversibly, the eIF2-bound GTP is hydrolysed, then 60S subunit joins the 48S complex, and elongation begins with complete 80S ribosome. We use two powerful approaches for visualization of 48S and 80S translation initiation complexes: primer extension-based (toe-printing) assay, and RelE-mediated mRNA hydrolysis (RelE-printing) which has been shown to occur exclusively in the A-site of the ribosome. Applying these two methods to cap-dependent initiation on beta-globin mRNA in the reconstituted translation assay, we detected transient intermediate complexes and monitored their sliding along mRNA toward the authentic AUG codon. Unexpectedly, when two close spaced AUG codon was present, in the conditions of blocked GTP hydrolysis, 48S pre-initiation complexes did not stopped irreversibly at the upstream codon but slid to the downstream one, although when GTP hydrolysis was allowed, 80S complex formed mainly at the upstream AUG. Since in living cells the rate of eIF2-bound GTP hydrolysis thought to be tightly regulated, this finding may be a key point in explaining the regulation of a start codon selection at mRNAs containing two or more close-spaced AUGs. Finally, using the toe-printing assay we compare kinetics of cap-dependent and IRES-dependent initiation and found out that the rate of 48S complex assembly is about an order of magnitude faster in the case of cap-dependent mRNAs.