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The role of arginine 244 in Candida glabrata tRNA nucleotidyltransferase

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The role of arginine 244 in Candida glabrata tRNA nucleotidyltransferase

Arthur, Jason (2009) The role of arginine 244 in Candida glabrata tRNA nucleotidyltransferase. Masters thesis, Concordia University.

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Abstract

The enzyme ATP(CTP):tRNA nucleotidyltransferase is essential in eukaryotes to allow tRNAs to participate in protein synthesis. It adds a specific cytidine-cytidine-adenosine (CCA) sequence to generate positions 74, 75, and 76 at the 3' ends of tRNAs in a nucleic acid template-independent manner. Considerable effort has gone into exploring the catalytic mechanism of this enzyme and several bacterial forms, as well as the human enzyme, have been crystallized either in the presence or absence of substrates. Here, we will discuss the role of an essential conserved arginine in a novel attempt to link in vitro enzyme activity to a measurable in vivo phenotype. We found that changing arginine 244 in Candida glabrata tRNA nucleotidyltransferase to alanine, lysine, or methionine resulted in cell death, even though the variant enzymes showed 50-70% activity as compared to the wild-type enzyme under standard in vitro assay conditions using a mixed population of tRNAs as substrate. More specific assays monitoring the incorporation of nucleotides into tRNA with known 3' ends demonstrated a loss of nucleotide addition at position 74, a reduction of nucleotide specificity at position 75, and an increase in nucleotide specificity at position 76 as compared to the native enzyme. These results suggest that arginine 244 in C. glabrata tRNA nucleotidyltransferase plays a role not only in binding and orienting the substrates (nucleotide triphosphates and tRNA) for catalysis but also in excluding ATP from the active site prior to nucleotide addition to positions 74 and 75.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (Masters)
Authors:Arthur, Jason
Pagination:xii, 99 leaves : ill. ; 29 cm.
Institution:Concordia University
Degree Name:M. Sc.
Program:Chemistry
Date:2009
Thesis Supervisor(s):Joyce, Paul
ID Code:976217
Deposited By: Concordia University Library
Deposited On:22 Jan 2013 16:21
Last Modified:18 Jan 2018 17:42
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