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The 2'-5' RNA Ligase of Escherichia coli: Purification, Cloning, and Genomic Disruption

Arn, Eric A. and Abelson, John N. (1996) The 2'-5' RNA Ligase of Escherichia coli: Purification, Cloning, and Genomic Disruption. Journal of Biological Chemistry, 271 (49). pp. 31145-31153. ISSN 0021-9258. http://resolver.caltech.edu/CaltechAUTHORS:ARNjbc96

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Abstract

An RNA ligase previously detected in extracts of Escherichia coli is capable of joining Saccharomyces cerevisiae tRNA splicing intermediates in the absence of ATP to form a 2-5 phosphodiester linkage (Greer, C., Javor, B., and Abelson, J. (1983) Cell 33, 899-906). This enzyme specifically ligates tRNA half-molecules containing nucleoside base modifications and shows a preference among different tRNA species. In order to investigate the function of this enzyme in RNA metabolism, the ligase was purified to homogeneity from E. coli lysate utilizing chromatographic techniques and separation of proteins by SDS-polyacrylamide gel electrophoresis. A single polypeptide of approximately 20 kilodaltons exhibited RNA ligase activity. The amino terminus of this protein was sequenced, and the open reading frame (ORF) encoding it was identified by a data base search. This ORF, which encodes a novel protein with a predicted molecular mass of 19.9 kDa, was amplified from E. coli genomic DNA and cloned. ORFs coding for highly similar proteins were detected in Methanococcus jannaschii and Bacillus stearothermophilus. The chromosomal gene encoding RNA ligase in E. coli was disrupted, abolishing ligase activity in cell lysates. Cells lacking ligase activity grew normally under laboratory conditions. However, moderate overexpression of the ligase protein led to slower growth rates and a temperature-sensitive phenotype in both wild-type and RNA ligase knockout strains. The RNA ligase reaction was studied in vitro using purified enzyme and was found to be reversible, indicating that this enzyme may perform cleavage or ligation in vivo.


Item Type:Article
Additional Information:©1996 by The American Society for Biochemistry and Molecular Biology, Inc. Received for publication, August 7, 1996, and in revised form, September 18, 1996. We gratefully acknowledge the contribution of unpublished genomic sequences by Drs. Keiko Takemoto and Hirotada Mori. This research was greatly assisted by scientific advice and guidance from Drs. Reinhard Rauhut, Jaime Arenas, Jeff Sampson, Peggy Saks, and Olke Uhlenbeck. Thanks to Dr. John Wagner and Christopher Trotta for critical reading of this manuscript and to Herbert J. Botts for his inspiration.
Subject Keywords:GENE; ENDONUCLEASE; BACTERIA; EXTRACT; TRANSCRIPTION; SYNTHETASE; EXPRESSION; SEQUENCES; INTRONS
Record Number:CaltechAUTHORS:ARNjbc96
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:ARNjbc96
Alternative URL:http://dx.doi.org//http://www.jbc.org/cgi/content/abstract/271/49/31145
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:3551
Collection:CaltechAUTHORS
Deposited By: Lindsay Cleary
Deposited On:14 Jun 2006
Last Modified:26 Dec 2012 08:55

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