ИСТИНА

About two third of mammalian mRNAs possess relatively long (~200–300 nt) GC-rich 5'-untranslated regions (5'-UTRs), while the others, in contrast, have unstructured 5'-UTRs ~30-50 nt long. Until now, mechanisms of translation initiation have been studied only on mRNAs with rather "simple" unstructured leaders such as -globin, reovirus mRNAs and some artificial templates. In particular, the most promising approach for studying molecular mechanisms of translational initiation is reconstruction of initiation complexes from individual components (ribosome subunits, Met-tRNA, purified initiation factors and mRNA) – so far it has been successfully applied only to a small set of mRNA species due to failure to assemble initiation complexes with mRNAs containing any GC-rich secondary structures in 5'-UTR (e.g. mRNA for beta-actin, hsp70, late adenovirus mRNA, and much more). Nevertheless, such mRNAs are successfully translated in cell lysates, so the question arises if there is any unknown initiation factor(s) in lysates allowing them to initiate translation. Here we report that the initiation of translation on such mRNAs does not require any additional components except canonical ones, but initiation factor eIF4B is needed in a rather greater amount then in the case of mRNAs with unstructured 5'-UTRs. For the first time, we have studied quantitative requirements of mRNAs for eIF4B and also for other components of the RNA helicase complex (eIF4A and eIF4F) in a real translation initiation system. To this end, a set of closely related RNA transcripts differed only in amount of base-paired nucleotides in their 5'-UTR has been used. eIF2 requirements have been analyzed as well and found to increase in the case of mRNAs with structured 5'-UTRs as well, allowing us to propose a model in which eIF2 assists the 40S subunit to bind to mRNA's 5'-leader and to scan toward the initiation codon. This difference in requirements for concentrations of the initiation factors suggests a possibility of differential translational regulation of cap-dependent mammalian mRNAs with base-paired, GC-rich, and unstructured, GC-poor, 5'-UTRs.