Methionyl-tRNA synthetase-induced conformational change of Escherichia coli tRNAfMet.
Ribonuclease T2, nuclease S1, and snake venom phosphodiesterase were used as a structural probe for investigation of the interaction between Escherichia coli tRNAfMet and methionyl-tRNA synthetase, and the cleavage sites were analyzed by a rapid sequencing gel electrophoresis of 5'-32P-labeled tRNA. Both endonucleases cleaved the D-loop of synthetase-bound tRNA much more extensively than that of the free tRNA. Positions of A14, G15, A22, and G23 in the D-loop and C35 in the anticodon of the synthetase-bound tRNA were more susceptible to RNase T2. The synthetase-bound tRNA was predominantly cleaved by nuclease S1 at position of G15, G19, G20, and G23 in the D-loop and G2 in the acceptor stem. In contrast, the synthetase-bound tRNA was more resistant to the 3'-exonuclease, snake venom phosphodiesterase, than was the free tRNA molecule. These results suggest conformational change of the tRNA by the synthetase binding which weakened tertiary interaction between the D-loop and T psi C-loop/extra-loop. Production of acid-soluble radioactivity was also examined in the limited digestion of 5'-32P-labeled tRNA or 3'-14C-labeled methionyl-tRNA. The synthetase enhanced the release of acid-soluble oligonucleotides from the 5'-end of the tRNA but suppressed that from the 3'-end of the molecule. These results are consistent with that obtained by gel electrophoresis.
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001483:Base Sequence; D004720:Endonucleases / Q000378:metabolism; D004722:Endoribonucleases; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008718:Methionine-tRNA Ligase / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012358:RNA, Transfer, Met; D012260:Ribonucleases / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases
D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D004720:Endonucleases; D004722:Endoribonucleases; D012260:Ribonucleases; C022543:ribonuclease T(2); D015719:Single-Strand Specific DNA and RNA Endonucleases; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase