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

J. Biol. Chem., Vol. 278, Issue 45, 44001-44008, November 7, 2003

This Article Full Text Full Text (PDF) All Versions of this Article: 278/45/44001    most recent M306760200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted 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 Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Redko, Y. Articles by McDowall, K. J. Search for Related Content PubMed PubMed Citation Articles by Redko, Y. Articles by McDowall, K. J. Social Bookmarking                      What's this?

Determination of the Catalytic Parameters of the N-terminal Half of Escherichia coli Ribonuclease E and the Identification of Critical Functional Groups in RNA Substrates^*

Yulia Redko, Mark R. Tock, Chris J. Adams, Vladimir R. Kaberdin¶||, Jane A. Grasby, and Kenneth J. McDowall**

From the Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Manton Building, LS2 9JT Leeds, United Kingdom, Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Dainton Building, Brook Hill, S3 7HF Sheffield, United Kingdom, and ^¶Max F. Perutz Laboratories, Department of Microbiology and Genetics, Vienna Biocenter, Dr. Bohrgasse 9, A-1030 Vienna, Austria

Ribonuclease E is required for the rapid decay and correct processing of RNA in Escherichia coli. A detailed understanding of the hydrolysis of RNA by this and related enzymes will require the integration of structural and molecular data with quantitative measurements of RNA hydrolysis. Therefore, an assay for RNaseE that can be set up to have relatively high throughput while being sensitive and quantitative will be advantageous. Here we describe such an assay, which is based on the automated high pressure liquid chromatography analysis of fluorescently labeled RNA samples. We have used this assay to optimize reaction conditions, to determine for the first time the catalytic parameters for a polypeptide of RNaseE, and to investigate the RNaseE-catalyzed reaction through the modification of functional groups within an RNA substrate. We find that catalysis is dependent on both protonated and unprotonated functional groups and that the recognition of a guanosine sequence determinant that is upstream of the scissile bond appears to consist of interactions with the exocyclic 2-amino group, the 7N of the nucleobase and the imino proton or 6-keto group. Additionally, we find that a ribose-like sugar conformation is preferred in the 5'-nucleotide of the scissile phosphodiester bond and that a 2'-hydroxyl group proton is not essential. Steric bulk at the 2' position in the 5'-nucleotide appears to be inhibitory to the reaction. Combined, these observations establish a foundation for the functional interpretation of a three-dimensional structure of the catalytic domain of RNaseE when solved.

Received for publication, June 25, 2003 , and in revised form, August 27, 2003.

^* This work was supported by a grant from the United Kingdom BBSRC (to K. J. M.). 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.

^|| Supported by Grant F1707 from the Austrian Science Foundation.

^** Recipient of a Royal Society University Research Fellowship. To whom correspondence should be addressed. Tel.: 44-113-343-3109; Fax: 44-113-343-2835; E-mail: k.j.mcdowall{at}leeds.ac.uk.

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

This article has been cited by other articles:

				J. M. Hurley and N. A. Woychik Bacterial Toxin HigB Associates with Ribosomes and Mediates Translation-dependent mRNA Cleavage at A-rich Sites J. Biol. Chem., July 10, 2009; 284(28): 18605 - 18613. [Abstract] [Full Text] [PDF]

				N. Beloglazova, G. Brown, M. D. Zimmerman, M. Proudfoot, K. S. Makarova, M. Kudritska, S. Kochinyan, S. Wang, M. Chruszcz, W. Minor, et al. A Novel Family of Sequence-specific Endoribonucleases Associated with the Clustered Regularly Interspaced Short Palindromic Repeats J. Biol. Chem., July 18, 2008; 283(29): 20361 - 20371. [Abstract] [Full Text] [PDF]

				X. Jiang and J. G. Belasco Catalytic activation of multimeric RNase E and RNase G by 5'-monophosphorylated RNA PNAS, June 22, 2004; 101(25): 9211 - 9216. [Abstract] [Full Text] [PDF]