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Purification of yeast transfer RNA ligase

Xu, Qi and Phizicky, Eric M. and Greer, Chris L. and Abelson, John N. (1990) Purification of yeast transfer RNA ligase. In: RNA processing. Part B, Specific methods. Methods in enzymology. No.181. Academic Press , San Diego, CA, pp. 463-471. ISBN 9780121820824. http://resolver.caltech.edu/CaltechAUTHORS:20131025-140808450

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Abstract

The splicing of yeast tRNA precursors requires the action of at least two proteins, an endonuclease which cleaves the two splice junctions to release the linear intron and a ligase which joins the resulting tRNA half molecules together. Each of the activities of tRNA ligase can be assayed independent of the overall reaction. Researchers have routinely assayed the enzyme activity during purification by the ligation of tRNA half-molecules produced by the action of tRNA endonuclease. The pre-tRNA phe substrate can now conveniently be synthesized in vitro by T7 RNA polymerase transcription of a synthetic gene. One microliter of each reaction mixture is applied to a cellulose plate which is developed in solvent containing saturated ammonium sulfate, 1 M sodium acetate, 2-propanol (40:9: 1) and then analyzed by autoradiography. The amounts of cyclic GMP and 2′-GMP are quantitated by cutting the spots from the thin-layer plate and counting them in a scintillation counter.


Item Type:Book Section
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http://www.sciencedirect.com/science/article/pii/007668799081144JPublisherArticle
Additional Information:© 1990 Academic Press, Inc.
Record Number:CaltechAUTHORS:20131025-140808450
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20131025-140808450
Official Citation:Qi Xu, Eric M. Phizicky, Chris L. Greer, John N. Abelson, Purification of yeast transfer RNA ligase, In: James E. Dahlberg, John N. Abelson, Editor(s), Methods in Enzymology, Academic Press, 1990, Volume 181, Pages 463-471, ISSN 0076-6879, ISBN 9780121820824, http://dx.doi.org/10.1016/0076-6879(90)81144-J. (http://www.sciencedirect.com/science/article/pii/007668799081144J)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:42078
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:25 Oct 2013 21:43
Last Modified:25 Oct 2013 21:43

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