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Analysis of the Cleavage Mechanism by Protein-Only RNase P Using Precursor tRNA Substrates with Modifications at the Cleavage Site
- Source :
- Journal of Molecular Biology. 428:4917-4928
- Publication Year :
- 2016
- Publisher :
- Elsevier BV, 2016.
-
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.
- Subjects :
- 0301 basic medicine
Cleavage factor
Arabidopsis Proteins
RNase P
Arabidopsis
Biology
Cleavage (embryo)
RNase PH
Catalysis
Ribonuclease P
Substrate Specificity
Kinetics
03 medical and health sciences
RNase MRP
030104 developmental biology
RNA, Transfer
Biochemistry
Structural Biology
Transfer RNA
Phosphodiester bond
RNA Precursors
Molecular Biology
Ribonucleoprotein
Subjects
Details
- ISSN :
- 00222836
- Volume :
- 428
- Database :
- OpenAIRE
- Journal :
- Journal of Molecular Biology
- Accession number :
- edsair.doi.dedup.....0dd16c76ba11bb9f482d507bc81c60b9