Role of base G-2 of pre-tRNAfMet in cleavage site selection by Escherichia coli RNase P in vitro

Biochemistry
M Lazard, T Meinnel

Abstract

In this study, a protocol for the purification of fully active Escherichia coli RNase P holoenzyme from a strain overproducing both the C5 protein and the M1 RNA components is described. A total of 0. 8 mg of homogeneous enzyme, with a 1:1 protein/RNA subunit stoichiometry, was recovered from a 1 L bacterial culture. In addition, a convenient and reliable method based on capillary gel electrophoresis was developed to measure initial rates of pre-tRNA maturation by RNase P. Using these tools, the kinetic parameters of cleavage by RNase P of various mutants of pre-tRNAfMet showing maturation defects in vivo [Meinnel and Blanquet (1995) J. Biol. Chem. 270, 15906-15914] were investigated in vitro and the locations of cleavage sites were determined from the length of the various products of the reaction. The nucleotide at position -2 of pre-tRNAfMet is shown to be important only in the selection of the cleavage site, whereas it has no role in the efficiency of the reaction. It is concluded that base G-2 acts as an antideterminant by preventing an alternative cleavage by RNase P. In addition, the presence of G-2 alone is enough to fully compensate for the lack of a G at position +1 of pre-tRNAfMet.

References

Oct 20, 1992·Journal of Molecular Biology·S G Svärd, L A Kirsebom
Aug 20, 1988·Journal of Molecular Biology·A VioqueS Altman
Oct 1, 1993·Science·Z TsuchihashiD Herschlag
Jan 1, 1993·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·S AltmanS Talbot

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Citations

Nov 30, 2002·Biochimie·Mathias BrännvallLeif A Kirsebom
Oct 20, 2009·Applied and Environmental Microbiology·Shoji Ohuchi, Dirk Schüler
Sep 5, 2012·Journal of Chromatography. a·Yi H NaiMichael C Breadmore
Feb 16, 2011·Methods : a Companion to Methods in Enzymology·Luc Ponchon, Frédéric Dardel
Feb 27, 2009·IUBMB Life·Leif A Kirsebom, Stefan Trobro
Oct 29, 1998·Journal of Molecular Biology·M BrännvallL A Kirsebom

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