title: tRNA processing by protein-only versus RNA-based RNase P: kinetic analysis reveals mechanistic differences.
abstract: In Arabidopsis thaliana, RNase P function, that is, endonucleolytic tRNA 5'-end maturation, is carried out by three homologous polypeptides ("proteinaceous RNase P" (PRORP) 1, 2 and 3). Here we present the first kinetic analysis of these enzymes. For PRORP1, a specificity constant (k(react)/K(m(sto))) of 3×10(6) M(-1) min(-1) was determined under single-turnover conditions. We demonstrate a fundamentally different sensitivity of PRORP enzymes to an Rp-phosphorothioate modification at the canonical cleavage site in a 5'-precursor tRNA substrate; whereas processing by bacterial RNase P is inhibited by three orders of magnitude in the presence of this sulfur substitution and Mg(2+) as the metal-ion cofactor, the PRORP enzymes are affected by not more than a factor of five under the same conditions, without significantly increased miscleavage. These findings indicate that the catalytic mechanism utilized by proteinaceous RNase P is different from that of RNA-based bacterial RNase P, taking place without a direct metal-ion coordination to the (pro-)Rp substituent. As Rp-phosphorothioate and inosine modification at all 26 G residues of the tRNA body had only minor effects on processing by PRORP, we conclude that productive PRORP-substrate interaction is not critically dependent on any of the affected (pro-)Rp oxygens or guanosine 2-amino groups.
authors.affiliation: Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, 35037 Marburg, Germany.
authors.forename: Liudmila V
authors.initials: LV
authors.lastname: Pavlova
authors.forename: Markus
authors.initials: M
authors.lastname: Gössringer
authors.forename: Christoph
authors.initials: C
authors.lastname: Weber
authors.forename: Aurélie
authors.initials: A
authors.lastname: Buzet
authors.forename: Walter
authors.initials: W
authors.lastname: Rossmanith
authors.forename: Roland K
authors.initials: RK
authors.lastname: Hartmann
mesh_terms: D017360:Arabidopsis / Q000201:enzymology*; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D054735:Phosphorothioate Oligonucleotides / Q000737:chemistry; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism*; D043262:Ribonuclease P / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D054735:Phosphorothioate Oligonucleotides; D012322:RNA Precursors; D012343:RNA, Transfer; D043262:Ribonuclease P