Interaction of bacterial initiation factor 2 with initiator tRNA.
Interaction of bacterial polypeptide chain initiation factor 2 (IF-2) with Escherichia coli formylmethionyl-tRNA in the absence of free Mg2+ renders the fMet-tRNA adsorable to nitrocellulose membrane filters. This reaction does not require GTP and is strongly inhibited by low concentration (1 mM) of Mg2+ in the reaction mixture. The structural requirements of the tRNA for binary complex formation have been studied using modified fMet-tRNAfMet molecules and a series of N-blocked and normal aminoacyl-rRNAs. It has been observed that IF-2 will not blind either to free formylmethionine or to a short fMet-oligonucleotide, but will bind to any xRNA structure covalently attached to an N-blocked methionine group. The E. coli initiator and noninitiator methiionine tRNAs, which have many differences in primary structure, were found to bind identically. In addition, fMet-tRNAfMet molecules containing structural modidifications at 20 different sites had the same affinity for IF-2 as unmodified fMet-tRNAfMet. N-blocked eukaryotic initiator tRNAs were also found to bind strongly to the factor. Binary complex formation was readily reversible, f[14C]Met-tRNAfMet being competed out by addition of an equal amount of unlabeled fMet-tRNAfMet to the preformed complex. In contrast, deacylated tRNAfMet was a poor compeitor, a 30-fold excess being required for 50% inhibition of complex formation in the presence of limiting factor. Although tRNAs having an N-blocked methionine were found to have the greatest affinity for IF-2, specificity for the amino acid in binary complex formation was not absolute. Partial binding was observed with N-substituted tyrosine, valine, and phenylalanine tRNAs, and weak or no binding with N-subsituted lysine, alanine, and leucine tRNAs. In all cases, N-blocked derivatives had a higher affinity for IF-2 than the corresponding unsubstituted aminoacyl-tRNAs. These results indicate that IF-2 alone is not capable of distinguishing the nucleotide sequence of tRNAs and selects the initiator tRNA by recognizing the fMet moiety. The overall data suggest that the role of IF-2 in formation of the ribosomal initiation complex is to stablize the interaction of fMet-tRNAfMet with the ribosome at low Mg2+ concentrations by binding to both the ribosomal particle and the fMet group of the tRNA.
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D000641:Ammonia / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008715:Methionine; D010445:Peptide Elongation Factors; D010448:Peptide Initiation Factors; D011485:Protein Binding; D012343:RNA, Transfer / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism; D013045:Species Specificity; D014443:Tyrosine
D046911:Macromolecular Substances; D010445:Peptide Elongation Factors; D010448:Peptide Initiation Factors; D014443:Tyrosine; D000641:Ammonia; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D008715:Methionine; D000604:Amino Acyl-tRNA Synthetases; D008274:Magnesium