The kinetics and specificity of cleavage by RNase P is mainly dependent on the structure of the amino acid acceptor stem.
Cleavage by RNase P of the tRNA(His precursor yields a mature tRNA with an 8 base pair amino acid acceptor stem instead of the usual 7 base pair stem. Here we show, both in vivo and in vitro, that this is mainly dependent on the primary structure and length of the acceptor stem in the precursor. Furthermore, the tRNA(His) precursor used in this study was processed with a change in both kinetic constants, Km and kcat, in comparison to the kinetics of cleavage of the precursor to tRNA(Tyr)Su3. Cleavage of a chimeric tRNA precursor showed that these altered kinetics were due to a difference in the primary structure and in the length of the acceptor stems of these two tRNA precursors. We also studied the cleavage reaction as a function of base substitutions at positions -1 and/or +73 in the precursor to tRNA(His). Our results suggest that the nucleotide at position +73 in tRNA(His) plays a significant role in the kinetics of cleavage of its precursor, possibly in product release. In addition, it appears that the C5 protein of RNase P is involved in the interaction between the enzyme and its substrate in a substrate-dependent manner, as previously suggested.
Department of Microbiology, Biomedical Center, Uppsala, Sweden.
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Kirsebom
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Svärd
D001482:Base Composition / Q000235:genetics; D001483:Base Sequence; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000378:metabolism*; D012329:RNA, Bacterial / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism*; D012354:RNA, Transfer, His / Q000378:metabolism*; D012365:RNA, Transfer, Tyr / Q000378:metabolism*; D043262:Ribonuclease P
D029968:Escherichia coli Proteins; D012322:RNA Precursors; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D012354:RNA, Transfer, His; D012365:RNA, Transfer, Tyr; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli