title: The exocyclic amine at the RNase P cleavage site contributes to substrate binding and catalysis.
abstract: Most tRNAs carry a G at their 5' termini, i.e. at position +1. This position corresponds to the position immediately downstream of the site of cleavage in tRNA precursors. Here we studied RNase P RNA-mediated cleavage of substrates carrying substitutions/modifications at position +1 in the absence of the RNase P protein, C5, to investigate the role of G at the RNase P cleavage site. We present data suggesting that the exocyclic amine (2NH2) of G+1 contributes to cleavage site recognition, ground state binding and catalysis by affecting the rate of cleavage. This is in contrast to O6, N7 and 2'OH that are suggested to affect ground state binding and rate of cleavage to significantly lesser extent. We also provide evidence that the effects caused by the absence of 2NH2 at position +1 influenced the charge distribution and conceivably Mg2+ binding at the RNase P cleavage site. These findings are consistent with models where the 2NH2 at the cleavage site (when present) interacts with RNase P RNA and/or influences the positioning of Mg2+ in the vicinity of the cleavage site. Moreover, our data suggest that the presence of the base at +1 is not essential for cleavage but its presence suppresses miscleavage and dramatically increases the rate of cleavage. Together our findings provide reasons why most tRNAs carry a guanosine at their 5' end.
authors.affiliation: Department of Cell and Molecular Biology, Uppsala University, Box 596, Biomedical Centre, SE-751 24 Uppsala, Sweden.
authors.forename: Ema
authors.initials: E
authors.lastname: Kikovska
authors.forename: Mathias
authors.initials: M
authors.lastname: Brännvall
authors.forename: Leif A
authors.initials: LA
authors.lastname: Kirsebom
mesh_terms: D000588:Amines / Q000737:chemistry*; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D006151:Guanosine / Q000737:chemistry*; D006878:Hydroxides / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D009584:Nitrogen / Q000737:chemistry; D009690:Nucleic Acid Conformation; D010100:Oxygen / Q000737:chemistry; D011485:Protein Binding; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics; D043262:Ribonuclease P / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D000588:Amines; D006878:Hydroxides; D006151:Guanosine; D012343:RNA, Transfer; D043262:Ribonuclease P; D008274:Magnesium; D009584:Nitrogen; D010100:Oxygen