A second tRNA binding site on elongation factor Tu is induced while the factor is bound to the ribosome.
Previously, we reported that the antibiotic kirromycin induces two tRNA-binding sites on the elongation factor Tu. The classical binding site (site I) binds aminoacyl-tRNA and, with much less affinity, deacylated tRNA. The kirromycin-induced site II binds aminoacyl-tRNA, peptidyl-tRNA, and deacylated tRNA with comparable affinities. Accordingly, 3'-oxidized tRNA can be cross-linked in the presence of the antibiotic to two specific sites of EF-Tu: Lys-237 and Lys-208. Here, we report that 3'-oxidized tRNAPhe, bound to a ribosome-poly(U) complex, can also be cross-linked to either one of these two sites. When located in the ribosomal peptidyl site, it cross-links exclusively to Lys-208; when located in the ribosomal aminoacyl site, it cross-links exclusively to Lys-237, irrespective of the presence of kirromycin. Since no cross-linking could be detected in the absence of ribosomes and kirromycin, we conclude that the tRNA-binding site II is induced upon interaction of aminoacyl-tRNA-EF-Tu-GTP with the ribosome-mRNA complex. The results indicate that, on the ribosome, EF-Tu interacts with peptidyl-site-bound peptidyl-tRNA through tRNA-binding site II and with aminoacyl-site-bound aminoacyl-tRNA through tRNA-binding site I.
D001665:Binding Sites; D003432:Cross-Linking Reagents; D004926:Escherichia coli; D008239:Lysine; D010444:Peptide Elongation Factor Tu; D010445:Peptide Elongation Factors / Q000378:metabolism*; D011072:Poly U / Q000235:genetics; D012343:RNA, Transfer / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism* / Q000648:ultrastructure
D003432:Cross-Linking Reagents; D010445:Peptide Elongation Factors; D011072:Poly U; D012343:RNA, Transfer; D010444:Peptide Elongation Factor Tu; D008239:Lysine