Effect of trypsin modification of the Escherichia coli elongation factor Tu on the ternary complex with aminoacyl-tRNA.
The ribonuclease resistance assay has been used to probe the effect of trypsin modification of the Escherichia coli elongation factor Tu X GTP on the interaction with E. coli aminoacyl-tRNAs. First, the equilibrium dissociation constant of the trypsin-modified Tu X GTP X Thr-tRNA complex was determined to be 2.3 (0.1) X 10(-5)M at 4 degrees C, pH 7.4. Second, binding of 17 of 20 noninitiator aminoacyl-tRNAs and four sets of purified isoacceptor tRNAs to the modified protein was measured. At 4 degrees C, the complex stabilities vary 500-fold over the range of aminoacyl-tRNAs, with Gln-tRNA forming the strongest ternary complex and Val-tRNA, the weakest. The results are compared to a similar study of ternary complex formation using intact elongation factor Tu X GTP, and the major differences are discussed. An analysis of both data sets, particularly that for the leucine isoacceptor tRNAs, suggests that the trypsin modification of elongation factor Tu X GTP disrupts a region of protein that is involved with the aminoacyl side chain rather than that of the acceptor stem helix region of the aminoacyl-tRNA.
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000032:analysis; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu; D010445:Peptide Elongation Factors; D012346:RNA, Transfer, Amino Acyl; D012260:Ribonucleases / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology*
D010445:Peptide Elongation Factors; D012346:RNA, Transfer, Amino Acyl; D006160:Guanosine Triphosphate; D012260:Ribonucleases; D014357:Trypsin; D010444:Peptide Elongation Factor Tu