HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 280, Issue 18, 17857-17862, May 6, 2005

This Article Full Text Full Text (PDF) All Versions of this Article: 280/18/17857    most recent M500591200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Schilling, O. Articles by Vogel, A. Search for Related Content PubMed PubMed Citation Articles by Schilling, O. Articles by Vogel, A. Social Bookmarking                      What's this?

Exosite Modules Guide Substrate Recognition in the ZiPD/ElaC Protein Family^*

Oliver Schilling, Bettina Späth¶, Brenda Kostelecky, Anita Marchfelder¶, Wolfram Meyer-Klaucke, and Andreas Vogel||

From the European Molecular Biology Laboratory Outstation Hamburg, Notkestrasse 85, 22603 Hamburg and ^¶Molekulare Botanik, Universität Ulm, 89069 Ulm, Germany

Escherichia coli ZiPD is the best characterized protein encoded by the elaC gene family and is a model for the 3'-pre-tRNA processing endoribonucleases (tRNase Z). A metal ligand-based sequence alignment of ZiPD with metallo--lactamase domain proteins of known crystallographic structure identifies a ZiPD-specific sequence insertion of 50 residues, which we will refer to as the ZiPD exosite. Functionally characterized ZiPD homologs from Bacillus subtilis, Methanococcus janaschii, and human share the presence of the ZiPD exosite, which is also present in the amino-terminal, but not in the carboxyl-terminal, domain of ElaC2 proteins. Another class of functionally characterized tRNase Z enzymes from Thermotoga maritima and Arabidopsis thaliana lack characteristic motifs in the exosite but possess a sequence segment with clustered basic amino acid residues. As an experimental attempt to investigate the function of the exosite we constructed a ZiPD variant that lacks this module (ZiPD). ZiPD has almost wild-type-like catalytic properties for hydrolysis of the small, chromogenic substrate bis(p-nitrophenyl) phosphate. Removal of the ZiPD exosite only affects k_cat, which is reduced by less than 40%, whereas both K' andthe Hill coefficient (measures of the substrate affinity and cooperativity, respectively) remain unchanged. Hence, the exosite is not required for the intrinsic phosphodiesterase activity of ZiPD. Removal of the exosite also does not affect the dimerization properties of ZiPD. In contrast to the wild-type enzyme, ZiPD does not process pre-tRNA, and gel shift assays demonstrate that only the wild-type enzyme, but not ZiPD, binds mature tRNA. These findings show that the exosite is essential for pre-tRNA recognition. In conclusion, we identify a ZiPD exosite that guides physiological substrate recognition in the ZiPD/ElaC protein family.

Received for publication, January 18, 2005 , and in revised form, February 7, 2005.

Addendum—After submission of this manuscript a report was published showing the crystal structure of the tRNase Z from B. subtilis (de la Sierra-Gallay, I. L., Pellegrini, O., and Condon, C. (2005) Nature 433, 657–661). The authors describe a flexible arm in the structure that is identical to the exosite we describe here. From a model with bound tRNA the authors propose a role of the exosite in substrate recognition, which perfectly complements our biochemical data.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

^|| To whom correspondence should be addressed: Max-Planck-Institut fuer Kohlenforschung, Kaiser-Wilhelm-Platz 1, D-45470 Muelheim/Ruhr, Germany. Tel.: 49-208-306-2387; Fax: 49-208-306-2985; E-mail: vogel{at}mpi-muelheim.mpg.de.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				A. Minagawa, H. Takaku, R. Ishii, M. Takagi, S. Yokoyama, and M. Nashimoto Identification by Mn2+ rescue of two residues essential for the proton transfer of tRNase Z catalysis Nucleic Acids Res., August 11, 2006; 34(13): 3811 - 3818. [Abstract] [Full Text] [PDF]

				N. Zareen, A. Hopkinson, and L. Levinger Residues in two homology blocks on the amino side of the tRNase Z His domain contribute unexpectedly to pre-tRNA 3' end processing RNA, June 1, 2006; 12(6): 1104 - 1115. [Abstract] [Full Text] [PDF]

				B. Kostelecky, E. Pohl, A. Vogel, O. Schilling, and W. Meyer-Klaucke The Crystal Structure of the Zinc Phosphodiesterase from Escherichia coli Provides Insight into Function and Cooperativity of tRNase Z-Family Proteins J. Bacteriol., February 15, 2006; 188(4): 1607 - 1614. [Abstract] [Full Text] [PDF]

				B. Spath, S. Kirchner, A. Vogel, S. Schubert, P. Meinlschmidt, S. Aymanns, J. Nezzar, and A. Marchfelder Analysis of the Functional Modules of the tRNA 3' Endonuclease (tRNase Z) J. Biol. Chem., October 21, 2005; 280(42): 35440 - 35447. [Abstract] [Full Text] [PDF]

				M. Ceballos-Chavez and A. Vioque Sequence-dependent Cleavage Site Selection by RNase Z from the Cyanobacterium Synechocystis sp. PCC 6803 J. Biol. Chem., September 30, 2005; 280(39): 33461 - 33469. [Abstract] [Full Text] [PDF]

				B. Ezraty, B. Dahlgren, and M. P. Deutscher The RNase Z Homologue Encoded by Escherichia coli elaC Gene Is RNase BN J. Biol. Chem., April 29, 2005; 280(17): 16542 - 16545. [Abstract] [Full Text] [PDF]