Study of the interaction of Escherichia coli methionyl-tRNA synthetase with tRNAfMet using chemical and enzymatic probes.
The accessibility of nucleotides in Escherichia coli tRNAfMet to chemical and enzymatic probes in the presence and absence of methionyl-tRNA synthetase has been investigated. Dimethyl sulfate was used to probe the reactivity of cytosine and guanosine residues. The N-3 position of the wobble anticodon base, C34, was strongly protected from methylation in the tRNA-synthetase complex. A synthetase-induced conformational change in the anticodon loop was suggested by the enhanced reactivity of C32 in the presence of enzyme. Cytosine residues in the dihydrouridine loop and in the 3'-terminal CCA sequence showed little or no change in reactivity. Methylation of the N-7 position of guanosine residues G42, G52, and G70 was partially inhibited by the synthetase. Nuclease digestion of tRNAfMet with alpha-sarcin in the presence of 1-2 mM Mg2+ resulted in cleavage mainly at C71 in the acceptor stem and was strongly inhibited by synthetase. Other nuclease digestion experiments using the single strand specific nucleases RNase A and RNase T1 revealed weak protection of nucleotides in the D loop and strong protection of nucleotides in the anticodon on complex formation. The present data, together with previous structure-function studies on this system, indicate strong binding of methionyl-tRNA synthetase to the anticodon of tRNAfMet, leading to a change in the conformation of the anticodon loop and stem. We propose that this, in turn, produces more distant, and possibly relatively subtle, conformational changes in other parts of the tRNA structure that ultimately lead to proper orientation of the 3' terminus of the tRNA with respect to the active site of the enzyme.
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001483:Base Sequence; D003562:Cytidine; D004926:Escherichia coli / Q000201:enzymology*; D006151:Guanosine; D007700:Kinetics; D008718:Methionine-tRNA Ligase / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D010761:Phosphorus Radioisotopes; D011485:Protein Binding; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012358:RNA, Transfer, Met; D006163:Ribonuclease T1; D012259:Ribonuclease, Pancreatic
D010761:Phosphorus Radioisotopes; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D006151:Guanosine; D003562:Cytidine; D006163:Ribonuclease T1; D012259:Ribonuclease, Pancreatic; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase