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Journal of Bacteriology, November 2007, p. 8339-8346, Vol. 189, No. 22
0021-9193/07/$08.00+0     doi:10.1128/JB.00713-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Young Kwan Kim,1,{dagger} Kenji Mizutani,3,{dagger} Kyung-Hee Rhee,1,{dagger} Ki-Hyun Nam,1 Won Ho Lee,1 Eun Hye Lee,1 Eunice Eunkyeong Kim,2 Sam-Yong Park,3 and Kwang Yeon Hwang1*

Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-701,1 Biomedical Research Center, Korea Institute of Science and Technology, Seoul 136-791, South Korea,2 Protein Design Laboratory, Yokohama City University, 1-7-29, Tsurumi, Yokohama 230-0045, Japan3

Received 4 May 2007/ Accepted 28 August 2007

In archaea, RNA endonucleases that act specifically on RNA with bulge-helix-bulge motifs play the main role in the recognition and excision of introns, while the eukaryal enzymes use a measuring mechanism to determine the positions 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 (EndATa) have been solved to 2.5-Å and 2.7-Å resolution by molecular replacement, using the 2.7-Å resolution data as the initial model and the single-wavelength anomalous-dispersion phasing method using selenomethionine as anomalous signals, respectively. The models show that EndATa is a homodimer and that it has overall folding similar to that of 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.

* Corresponding author. Mailing address: Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, South Korea. Phone: 82-2-3290-3009. Fax: 82-2-923-3225. E-mail: park{at}tsurumi.yokohama-cu.ac.jp or chahong{at}korea.ac.kr

{triangledown} Published ahead of print on 7 September 2007.

{dagger} Y.K.K., K.M., and K.H.R. contributed equally to this work.

Journal of Bacteriology, November 2007, p. 8339-8346, Vol. 189, No. 22
0021-9193/07/$08.00+0     doi:10.1128/JB.00713-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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