title: Analysis of the Cleavage Mechanism by Protein-Only RNase P Using Precursor tRNA Substrates with Modifications at the Cleavage Site.
abstract: Ribonuclease P (RNase P) is the enzyme that endonucleolytically removes 5'-precursor sequences from tRNA transcripts in all domains of life. RNase P activities are either ribonucleoprotein (RNP) or protein-only RNase P (PRORP) enzymes, raising the question about the mechanistic strategies utilized by these architecturally different enzyme classes to catalyze the same type of reaction. Here, we analyzed the kinetics and cleavage-site selection by PRORP3 from Arabidopsis thaliana (AtPRORP3) using precursor tRNAs (pre-tRNAs) with individual modifications at the canonical cleavage site, with either Rp- or Sp-phosphorothioate, or 2'-deoxy, 2'-fluoro, 2'-amino, or 2'-O-methyl substitutions. We observed a small but robust rescue effect of Sp-phosphorothioate-modified pre-tRNA in the presence of thiophilic Cd2+ions, consistent with metal-ion coordination to the (pro-)Sp-oxygen during catalysis. Sp-phosphorothioate, 2'-deoxy, 2'-amino, and 2'-O-methyl modification redirected the cleavage mainly to the next unmodified phosphodiester in the 5'-direction. Our findings are in line with the 2'-OH substituent at nucleotide -1 being involved in an H-bonding acceptor function. In contrast to bacterial RNase P, AtPRORP3 was found to be able to utilize the canonical and upstream cleavage site with similar efficiency (corresponding to reduced cleavage fidelity), and the two cleavage pathways appear less interdependent than in the bacterial RNA-based system.
authors: Institute of Pharmaceutical Chemistry, Philipps-University Marburg, 35037 Marburg, Germany.
authors: Dennis
authors: D
authors: Walczyk
authors: Institute of Pharmaceutical Chemistry, Philipps-University Marburg, 35037 Marburg, Germany.
authors: Markus
authors: M
authors: Gößringer
authors: Center for Anatomy & Cell Biology, Medical University of Vienna, 1090 Vienna, Austria.
authors: Walter
authors: W
authors: Rossmanith
authors: Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia; Skolkovo Institute of Science and Technology, 3 Nobel street, Innovation Center "Skolkovo", 143026 Skolkovo, Russia.
authors: Timofei S
authors: TS
authors: Zatsepin
authors: Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia.
authors: Tatiana S
authors: TS
authors: Oretskaya
authors: Institute of Pharmaceutical Chemistry, Philipps-University Marburg, 35037 Marburg, Germany. Electronic address: roland.hartmann@staff.uni-marburg.de.
authors: Roland K
authors: RK
authors: Hartmann
mesh_terms: D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D002384:Catalysis; D007700:Kinetics; D012322:RNA Precursors / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D043262:Ribonuclease P / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D029681:Arabidopsis Proteins; D012322:RNA Precursors; D012343:RNA, Transfer; C000609578:PRORP3 protein, Arabidopsis; D043262:Ribonuclease P
keywords: catalytic mechanism; phosphorothioate and 2′-ribose modifications at the canonical cleavage site; tRNA processing by protein-only RNase P PRORP3 from Arabidopsis thaliana