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. 1981 Mar;78(3):1542–1546. doi: 10.1073/pnas.78.3.1542

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

Raymond Kaempfer 1,*, John van Emmelo 1, Walter Fiers 1
PMCID: PMC319167  PMID: 6940171

Abstract

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 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 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.

Keywords: mRNA recognition, translation, RNA conformation, partial nuclease digestion

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Selected References

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