Specific binding of eukaryotic initiation factor 2 to satellite tobacco necrosis virus RNA at a 5′-terminal sequence comprising the ribosome binding site

Publisher Website
Google Scholar
Abstract
The mRNA-binding property of (rabbit reticulocyte) eukaryotic initiation factor 2 (eIF-2) was examined by studying its interaction with satellite tobacco necrosis virus (STNV) RNA carrying a 32P-labeled 5'' end. The RNA molecules bound by limiting amounts of eIF-2 were isolated and digested with pancreatic and T1 RNases. Digestion patterns showed that the labeled STNV RNA preparation offered to eIF-2 was heterogeneous, containing > 30 different 5'' ends; the RNA selected by eIF-2 possessed predominantly one 5'' end, pApGpUp..., the 5''-terminal sequence of intact STNV RNA. Binding analysis of individual 5''-terminal fragments generated from isolated, intact, STNV RNA by partial digestion with T1 RNase showed that eIF-2 does not bind detectably to the 32-nucleotide fragment ending with the initiation codon AUG or to shorter ones, but it does bind the 44 nucleotide fragment that contains the ribosome binding site. In addition to the structural features localized at the 5'' end of STNV RNA, eIF-2 appears to recognize a conformation found only in larger molecules, because intact RNA and large 5''-terminal fragments are bound preferentially over smaller ones. Binding of short 5''-terminal STNV RNA fragments to eIF-2 is specific, as judged by competition with STNV and rRNA. Binding of eIF-2 to intact STNV RNA leads to a conformational change in the RNA that greatly facilitates cleavage by T1 and P1 RNases at sites in the vicinity of the initiation region. eIF-2 thus interacts specifically with the 5''-terminal region of STNV RNA that contains the ribosome binding site and causes local unfolding of the RNA structure.