University of Illinois Urbana-Champaign

Biochemical and Structures Studies of tRNA Modificaton and Repair Enzymes

Zhou, Chun

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Description

Title
Biochemical and Structures Studies of tRNA Modificaton and Repair Enzymes
Author(s)
Zhou, Chun
Issue Date
2009
Doctoral Committee Chair(s)
Huang, Raven H.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
  • Chemistry
  • Biochemistry
Language
eng
Abstract
Plant siRNAs and miRNAs are methylated at their 3' ends at the 2' OH position by Hen1, an S-Adomet dependent methyltransferase. Hen1 homologs in animals are shown to be responsible for the methylation of piRNAs, some siRNAs as well as endogenous siRNAs. Interestingly, a subset of bacteria also has a Hen1 homolog. Since bacteria do not have canonical RNAi, Hen1 in bacteria (bHen1) might play other biological roles, Our goals are to find out the biological function of bHen1. We observed that immediately following bHen1, there was a second highly conserved gene (Pnkp) in the operon. Pnkp has previously been shown to have kinase, phosphatase and adenylyltransferase activity. Therefore Pnkp/Hen1 might be involved in RNA repair. I cloned and expressed both proteins with a pETDuet vector. Pnkp and bHen1 form a heterotetramer in gel filtration column. Purfied Pnkp/bHEN1 efficiently repaired full length tRNAs cleaved by bacterial toxins (colicin E5 and D). Before the broken RNAs were ligated, a methyl group was added to the 2' OH group that participated in the original RNA cut. Due to this methylation, RNAs repaired by bacterial Pnkp/Hen1 could not be cleaved again by the ribotoxins. Thus, unlike eukaryotic Hen1 involved in RNAi, bacterial Hen1 is part of a RNA repair and modification system.
Graduation Semester
2009
Type of Resource
text
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http://hdl.handle.net/2142/84867

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