Specific binding of eukaryotic initiation factor 2 to satellite tobacco necrosis virus RNA at a 5'-terminal sequence comprising the ribosome binding site.
The mRNA-binding property of eukaryotic initiation factor 2 (eIF-2) was examined by studying its interaction with satellite tobacco necrosis virus (STNV) RNA carrying a (32)P-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 more than 30 different 5' ends; by contrast, 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. However, binding of short 5'-terminal STNV RNA fragments to eIF-2 is specific, as judged by competition with STNV and ribosomal RNA. Finally, 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. These results show that eIF-2 interacts specifically with the 5'-terminal region of STNV RNA that contains the ribosome binding site and causes local unfolding of the RNA structure.
D000818:Animals; D001483:Base Sequence; D015852:Eukaryotic Initiation Factor-2; D007700:Kinetics; D009690:Nucleic Acid Conformation; D010179:Pancreas / Q000201:enzymology; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010942:Plant Viruses / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012260:Ribonucleases
D015852:Eukaryotic Initiation Factor-2; D010448:Peptide Initiation Factors; D011506:Proteins; D012367:RNA, Viral; D012260:Ribonucleases