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The role of arginine 204 in Candida glabrata tRNA nucleotidyltransferase

Title:

The role of arginine 204 in Candida glabrata tRNA nucleotidyltransferase

Colasurdo, Gabriele (2011) The role of arginine 204 in Candida glabrata tRNA nucleotidyltransferase. Masters thesis, Concordia University.

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Abstract

The enzyme ATP(CTP):tRNA nucleotidyltransferase is required for protein synthesis in eukaryotes. It allows for the step-wise addition of a specific cytidine-cytidine-adenosine (CCA) sequence to the 3’ ends of tRNAs without the use of a nucleic acid template. Crystal structures of the eubacterial and archeal enzymes have been solved both in the presence and absence of model substrates. Based on these studies and primary sequence comparisons, roles for a number of conserved residues have been proposed. Here, we examine the role of an arginine residue in the conserved EDxxR motif. In bacteria, this residue helps nucleotide selection by altering its orientation in space to make distinct hydrogen bonds first with CTP and then with ATP as part of a dynamic amino acid template (Li et al., 2002). We found that changing this arginine (Arg204) in the C. glabrata enzyme to alanine, glutamate, or glutamine, results in variant enzymes unable to support in vivo growth. Although biophysical experiments show differences between native and variant enzymes, Arg204’s primary role is not in defining the enzyme’s overall structural integrity. As expected, in vitro nucleotide incorporation experiments show a decrease in nucleotide incorporation efficiency at all positions, decreased specificity at position 75, and an increase in specificity at position 76 (compared to the native enzyme). Along with its suggested dynamic role during nucleotide binding, the results shown here suggest that Arg204 in C. glabrata tRNA nucleotidyltransferase also plays a role in orienting residues in the binding pocket while altering the pocket’s size to aid in discrimination between nucleotides at the different positions.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (Masters)
Authors:Colasurdo, Gabriele
Institution:Concordia University
Degree Name:M. Sc.
Program:Chemistry
Date:March 2011
Thesis Supervisor(s):Joyce, Paul
ID Code:7255
Deposited By:GABRIELE COLASURDO
Deposited On:13 Jun 2011 09:12
Last Modified:13 Jun 2011 09:12
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