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J. Bacteriol. doi:10.1128/JB.00713-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Structural and Mutational Analysis of tRNA-Intron Splicing Endonuclease from Thermoplasma acidophilum DSM 1728: The Catalytic Mechanism of tRNA-Intron Splicing Endonucleases

Division of Biotechnology, College of Life Sciences & Biotechnology, Korea University, Seoul 136-701, Biomedical Research Center, Korea Institute of Science and Technology, Seoul 136-791, South Korea, Protein Design Lab., Yokohama City University, 1-7-29 Seuhiro-cho, Tsurumi, Yokohama 230-0045, Japan

^* To whom correspondence should be addressed. Email: park{at}tsurumi.yokohama-cu.ac.jp. chahong{at}korea.ac.kr.

	Abstract

In archaea, RNA endonucleases which act specifically on RNA having bulge-helix-bulge (BHB) motifs play the main role in the recognition and excision of introns while the eukaryal enzymes use a measuring mechanism to determine the position of the universally positioned splice sites relative to the conserved domain of pre-tRNA. Two crystallographic structures of tRNA-intron splicing endonuclease from Thermoplasma acidophilum DSM 1728 (TaEndA) have been solved to 2.5 Å and 2.7 Å resolution by molecular replacement using the 2.7 Å resolution data as initial model and single wavelength anomalous dispersion phasing method using selenomethionine as anomalous signals, respectively. The models show that the TaEndA is a homodimer and has similar overall folding to other archaeal tRNA endonucleases. From structural and mutational analyses of H236A, Y229F, and K265I, in vitro, we have demonstrated that they play critical roles in recognizing the splice site and in cleaving the pre-tRNA substrate.