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Journal of Biochemistry Advance Access originally published online on November 13, 2008
Journal of Biochemistry 2009 145(2):151-160; doi:10.1093/jb/mvn150
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© The Authors 2008. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Analysis on Substrate Specificity of Escherichia coli Ribonuclease P Using Shape Variants of pre-tRNA: Proposal of Subsites Model for Substrate Shape Recognition

Satoshi Suwa, Yasuhiro Nagai, Akihiro Fujimoto, Yo Kikuchi and Terumichi Tanaka*

Division of Bioscience and Biotechnology, Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi, Aichi 441-8580, Japan

*To whom correspondence may be addressed. Tel: +81-53-244-6920, Fax: +81-53-244-6929, E-mail: tanakat{at}eco.tut.ac.jp

Received October 2, 2008; Accepted October 29, 2008


  Abstract

We prepared a series of shape variants of a pre-tRNA and examined substrate shape recognition by bacterial RNase P ribozyme and holoenzyme. Cleavage site analysis revealed two new subsites for accepting the T-arm and the bottom half of pre-tRNA in the substrate-binding site of the enzyme. These two subsites take part in cleavage site selection of substrate by the enzyme: the cleavage site is not always selected according to the relative position of the 3'-CCA sequence of the substrate. Kinetic studies indicated that the substrate shape is recognized mainly in the transition state of the reaction, and neither the shape nor position of either the T-arm or the bottom half of the substrate affected the Michaelis complex formation. These results strongly suggest that the 5' and 3' termini of a substrate are trapped by the enzyme first, then the position and the shape of the T-arm and the bottom half are examined by the cognate subsites. From these facts, we propose a new substrate recognition model that can explain many experimental facts that have been seen as enigmatic.

Key Words: C5 protein, M1 RNA, ribozyme, RNase P, subsite

Abbreviations: RNase P, ribonuclease P; pre-tRNA, tRNA precursor


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