Effect of the amino acid attached to Escherichia coli initiator tRNA on its affinity for the initiation factor IF2 and on the IF2 dependence of its binding to the ribosome.
We show that the nature of the amino acid in the formylaminoacyl-tRNA influences initiation factor (IF) 2 dependence of its ribosome binding and that this IF2 dependence reflects the relative affinity of the formylaminoacyl-tRNA for the initiation factor IF2. We compared the template-dependent ribosome binding activities, in the presence of initiation factors, of wild type and anticodon sequence mutants of Escherichia coli initiator tRNAs that carry formylmethionine (fMet), formylglutamine (fGln), or formylvaline (fVal). The fGln-tRNA bound less well than fMet-tRNA whereas the fVal-tRNA bound as well as fMet-tRNA. The rate and extent of binding of fGln-tRNA to the ribosome was significantly increased by further addition of purified initiation factor IF2. In contrast, the binding of fVal-tRNA or fMet-tRNA was not affected much by the addition of IF2. Using gel mobility shift assay, we have measured the apparent Kd values of the IF2.formylaminoacyl-tRNA binary complexes. These are 1.8, 3.5, and 10.5 microM for fMet-tRNA, fVal-tRNA, and fGln-tRNA, respectively.
Wu
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Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
RajBhandary
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D000596:Amino Acids / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012329:RNA, Bacterial / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*
D000596:Amino Acids; D015852:Eukaryotic Initiation Factor-2; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; D012343:RNA, Transfer