The 2'-5' RNA ligase of Escherichia coli : purification, cloning, and investigations of in vivo function

Citation

Arn, Eric (1996) The 2'-5' RNA ligase of Escherichia coli : purification, cloning, and investigations of in vivo function. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/d36x-7p54. https://resolver.caltech.edu/CaltechETD:etd-09122006-130201

Abstract

An RNA ligase activity has previously been detected in extracts of E. coli which is capable of joining S. cerevisiae tRNA splicing intermediates in the absence of ATP to form a 2'-5'phosphodiester linkage (36). In order to study the mechanism and function of this unusual enzyme in bacterial RNA metabolism, a purification of the ligase enzyme was undertaken. The ligase was purified to homogeneity from a soluble high-speed extract of E. coli utilizing standard chromatographic techniques and reconstitution of activity following separation by SDS-PAGE. A single active polypeptide of approximately 20 kiloDaltons (kD) was shown to provide RNA ligase activity. This protein was N-terminally sequenced, and the open reading frame (ORF) encoding it was identified by a database search. This ORF, which codes for a novel protein with a predicted molecular weight of 19.9 kD, was cloned by PCR and used for overexpression of active recombinant lipase in E. coli and S. cerevisiae. The single chromosomal gene encoding the ligase was disrupted by insertion, abolishing ligase activity. Cells lacking active ligase are viable and show growth kinetics identical to the parent strain. Either parent strain or ligase knockout expressing high levels of recombinant ligase grow slowly compared to wild type and are temperature sensitive. Computer analysis of the ligase protein sequence allowed prediction of antigenic peptides derived from it. These peptides were synthesized and injected into rabbits to elicit polyclonal anti-ligase antibodies. Such antibodies were purified from rabbit sera by affinity to immobilized ligase, and shown to specifically recognize the ligase protein on Western blots. Ligase minus strains, purified recombinant ligase, and anti-ligase antibodies have been utilized in a variety of experiments to attempt to identify the in vivo substrate of this enzyme.

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