The 2'-5' RNA ligase of Escherichia coli. Purification, cloning, and genomic disruption.
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.
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Division of Biology 147-75, California Institute of Technology, Pasadena, California 91125, USA. zoltan@BCMP.med.harvard.edu
Abelson
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D010641:Phenotype; D011130:RNA Ligase (ATP) / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013696:Temperature
D011130:RNA Ligase (ATP)