Nucleic Acids Research Advance Access originally published online on May 8, 2007
Nucleic Acids Research 2007 35(11):3624-3630; doi:10.1093/nar/gkm110
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Nucleic Acids Research, 2007, Vol. 35, No. 11 3624-3630
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Reprogramming the tRNA-splicing activity of a bacterial RNA repair enzyme
Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA
*To whom correspondence should be addressed. +1-212 639-7145; +1-212 717-3623; Email: s-shuman{at}ski.mskcc.org
Received January 26, 2007. Revised February 7, 2007. Accepted February 7, 2007.
Programmed RNA breakage is an emerging theme underlying cellular responses to stress, virus infection and defense against foreign species. In many cases, site-specific cleavage of the target RNA generates 2',3' cyclic phosphate and 5'-OH ends. For the damage to be repaired, both broken ends must be healed before they can be sealed by a ligase. Healing entails hydrolysis of the 2',3' cyclic phosphate to form a 3'-OH and phosphorylation of the 5'-OH to form a 5'-PO4. Here, we demonstrate that a polynucleotide kinase-phosphatase enzyme from Clostridium thermocellum (CthPnkp) can catalyze both of the end-healing steps of tRNA splicing in vitro. The route of tRNA repair by CthPnkp can be reprogrammed by a mutation in the 3' end-healing domain (H189D) that yields a 2'-PO4 product instead of a 2'-OH. Whereas tRNA ends healed by wild-type CthPnkp are readily sealed by T4 RNA ligase 1, the H189D enzyme generates ends that are spliced by yeast tRNA ligase. Our findings suggest that RNA repair enzymes can evolve their specificities to suit a particular pathway.
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