Photochemical Cross-Linking of Cap Binding Proteins to Eucaryotic mRNAs: Effect of mRNA 5′ Secondary Structure

Abstract

We used UV light-induced cross-linking to study the interactions of cap binding proteins with the 5' cap structure of eucaryotic mRNAs. Thymidine kinase gene (herpes simplex virus type 1) transcripts prepared in vitro using the SP6 RNA polymerase transcription system were capped and methylated posttranscriptionally with [alpha-32P]GTP and S-adenosyl-L-methionine to yield cap-labeled transcripts. Irradiation of capped transcripts with crude rabbit reticulocyte initiation factors in the presence of ATP-Mg2+ resulted in the cap-specific cross-linking of two polypeptides with molecular masses of 24 and 80 kilodaltons (kDa). The cross-linking characteristics of these polypeptides resemble those of the cap-binding proteins previously detected by a chemical cross-linking assay (N. Sonenberg, D. Guertin, D. Cleveland, and H. Trachsel, Cell 27:563-572, 1981). However, the relative efficiency of the cross-linking of these two polypeptides to the cap structure was different from that in previous studies, and there was no detectable cross-linking of the previously described 50-kDa polypeptide. In addition, we present data indicating that the insertion of secondary structure into the 5' noncoding region of tk mRNA, 6 nucleotides from the cap structure, decreases the cap-specific cross-linking of the 80-kDa but not the 24-kDa polypeptide. In contrast, the insertion of secondary structure 37 nucleotides from the cap structure had no significant effect on the cross-linking of either the 24- or the 80-kDa cap-specific polypeptide. These results demonstrate that the position of mRNA 5'-proximal secondary structure relative to the cap structure can influence the cap-specific interaction between the mRNA and a translation initiation factor.