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 13:12 |
| Last Modified: | 18 Jan 2018 17:30 |
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