title: Several regions of a tRNA precursor determine the Escherichia coli RNase P cleavage site.
abstract: The RNase P cleavage reaction was studied as a function of the number of base-pairs in the acceptor-stem and/or T-stem of a natural tRNA precursor, the tRNA(Tyr)Su3 precursor. Our data suggest that the location of the Escherichia coli RNase P cleavage site does not depend merely on the lengths of the acceptor-stem and T-stem as previously suggested. Surprisingly, we find that precursors with only four base-pairs in the acceptor-stem are cleaved by M1 RNA and by holoenzyme. Furthermore, we show that both disruption of base-pairing, and alteration of the nucleotide sequence (without disruption of base-pairing) proximal to the cleavage site result in aberrant cleavage. Thus, the identity of the nucleotides near the cleavage site is important for recognition of the cleavage site rather than base-pairing. The important nucleotides are those at positions -2, -1, +1, +72, +73 and +74. We propose that the nucleotide at position +1 functions as a guiding nucleotide. These results raise the possibility that Mg2+ binding near the cleavage site is dependent on the identity of the nucleotides at these positions. In addition, we show that disruption of base-pairing in the acceptor-stem affects both Michaelis-Menten constants, Km and kcat.
mesh_terms: D001482:Base Composition; D001483:Base Sequence; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012329:RNA, Bacterial / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism*; D012365:RNA, Transfer, Tyr / Q000737:chemistry / Q000378:metabolism*; D043262:Ribonuclease P
chemical_list: D029968:Escherichia coli Proteins; D012322:RNA Precursors; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D012365:RNA, Transfer, Tyr; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli