Decoding at the ribosomal A site. The effect of a defined codon-anticodon mismatch upon the behavior of bound aminoacyl transfer RNA.
Ribosomes from Escherichia coli were programmed by being allowed to bind a molecule of tRNAMetf or fMet-tRNAMetf and the hexanucleotide messenger AUGN1N2N3. The interaction of the ternary complex [EF-Tu X GTP X Phe-tRNAPhe] with the A site (containing the codon N1N2N3) was then studied by measuring the extent of (i) the binding of Phe-tRNAPhe to the ribosome, (ii) the hydrolysis of GTP, and (iii) the formation of the dipeptide fMet-Phe. By variation of N1,N2, and N3, a defined degree and position of mismatch could be obtained; the correct A-site codon UUU was compared with the incorrect codons CUU, UCU, GUU, and UUG. Each single-point alteration led to catalytic hydrolysis of GTP and to a strong reduction in the amounts of Phe-tRNAPhe binding and of dipeptide formation. The observations were explicable qualitatively by a hypothesis according to which the behavior of the bound aa-tRNA, after hydrolysis of GTP and before peptidyl transfer, is determined principally by the energy of binding of the aminoacyl-tRNA to the A site. This binding in turn was found to depend upon both the nature and the position of the mismatch. The results further suggest a steric interplay between the 3' (acceptor) end of the A-site tRNA and the second and third positions of the anticodon, so that a mismatch at one of these positions can impair directly the interaction between the aminoacylated 3' end and the ribosome and can thus reduce the rate of peptide bond formation and contribute to the overall fidelity of the elongation cycle.
D000926:Anticodon / Q000378:metabolism*; D001483:Base Sequence; D003062:Codon / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D010444:Peptide Elongation Factor Tu; D010445:Peptide Elongation Factors / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012358:RNA, Transfer, Met; D012270:Ribosomes / Q000378:metabolism*
D000926:Anticodon; D003062:Codon; D010445:Peptide Elongation Factors; D012333:RNA, Messenger; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D010444:Peptide Elongation Factor Tu