NAIM and site-specific functional group modification analysis of RNase P RNA: magnesium dependent structure within the conserved P1-P4 multihelix junction contributes to catalysis

Biochemistry
Nicholas M KayeMichael E Harris

Abstract

The tRNA processing endonuclease ribonuclease P contains an essential and highly conserved RNA molecule (RNase P RNA) that is the catalytic subunit of the enzyme. To identify and characterize functional groups involved in RNase P RNA catalysis, we applied self-cleaving ribozyme-substrate conjugates, on the basis of the RNase P RNA from Escherichia coli, in nucleotide analogue interference mapping (NAIM) and site-specific modification experiments. At high monovalent ion concentrations (3 M) that facilitate protein-independent substrate binding, we find that the ribozyme is largely insensitive to analogue substitution and that concentrations of Mg2+ (1.25 mM) well below that necessary for optimal catalytic rate (>100 mM) are required to produce interference effects because of modification of nucleotide bases. An examination of the pH dependence of the reaction rate at 1.25 mM Mg2+ indicates that the increased sensitivity to analogue interference is not due to a change in the rate-limiting step. The nucleotide positions detected by NAIM under these conditions are located exclusively in the catalytic domain, consistent with the proposed global structure of the ribozyme, and predominantly occur within the highly conserved P1-P4 mult...Continue Reading

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Citations

Jan 1, 2003·Journal of Molecular Biology·Hsin-Yue TsaiVenkat Gopalan
Jul 2, 2003·Current Opinion in Structural Biology·Michael E Harris, Eric L Christian
Nov 15, 2002·Methods : a Companion to Methods in Enzymology·Eric L ChristianMichael E Harris
Sep 1, 2004·RNA·Yong Li, Sidney Altman
Jun 25, 2004·Bioscience, Biotechnology, and Biochemistry·Terumichi TanakaYo Kikuchi
Aug 5, 2009·Methods : a Companion to Methods in Enzymology·John K Frederiksen, Joseph A Piccirilli
Feb 27, 2009·IUBMB Life·Leif A Kirsebom, Stefan Trobro
Jan 13, 2005·Journal of Molecular Biology·Nathan H ZahlerMichael E Harris
Apr 28, 2006·Journal of Molecular Biology·Ema KikovskaLeif A Kirsebom
Nov 26, 2009·Journal of Molecular Biology·Kristin S KoutmouCarol A Fierke
Jan 27, 2010·Journal of Molecular Biology·William H McClainVenkat Gopalan
Apr 23, 2008·Current Protocols in Nucleic Acid Chemistry·Jesse C Cochrane, Scott A Strobel
Aug 21, 2008·Bioconjugate Chemistry·Alexander Berchanski, Aviva Lapidot

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