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Biophysical and biochemical characterization of yeast tRNA nucleotidyltransferase variants

Title:

Biophysical and biochemical characterization of yeast tRNA nucleotidyltransferase variants

Rahman, Mohammed Samiur (2017) Biophysical and biochemical characterization of yeast tRNA nucleotidyltransferase variants. Masters thesis, Concordia University.

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Abstract

The enzyme ATP(CTP):tRNA-specific tRNA nucleotidyltransferase adds cytidine-cytidine-adenosine (CCA) to the 3’ end of eukaryotic tRNAs during their maturation. This CCA sequence plays a vital role in aminoacylation and hence in protein synthesis. In yeast, this enzyme is defined as a Class II tRNA nucleotidyltransferase due to the presence of five conserved N-terminal motifs (A to E). Based on the available crystal structures of related tRNA nucleotidyltransferases, specific functions have been assigned to each of these motifs. We previously have shown that mutations in motif C that reduce enzyme activity can be overcome by a mutation in motif A that restores this activity. Here we explore the roles of two acidic residues (glutamate 189 and aspartate 190) found within motif C and one residue (arginine 64) found in motif A to understand better the role of motif C and the potential interactions between motifs A and C.

Site-directed mutagenesis was used to change arginine 64 (to tryptophan), or glutamate 189 (to glutamine, lysine, alanine or phenylalanine) or aspartate 190 (to alanine or phenylalanine) alone, or in combination with the arginine 64 tryptophan substitution and the effects of these amino acid alterations on enzyme structure and function were studied. Biophysical analyses (circular dichroism and fluorescence spectroscopy and thermal denaturation experiments) suggest no major changes in structure in almost all of the variants tested. Kinetic analysis revealed no alterations in substrate binding (Km), but a large drop in turnover number (kcat) for the 189 and 190 variants (but not the arginine 64 variant). The reduced activity in the 189 and 190 variants is alleviated when accompanied by the change of arginine 64 to tryptophan, which also suppresses the temperature-sensitive phenotype. Taken together these data suggest that arginine 64 is not required for enzyme activity unlike glutamate 189 and aspartate 190. Moreover, they suggest an interaction between motifs A and C, and that motif C plays a role in accommodating and orienting the substrates to promote catalysis involving motif A.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (Masters)
Authors:Rahman, Mohammed Samiur
Institution:Concordia University
Degree Name:M. Sc.
Program:Chemistry
Date:December 2017
Thesis Supervisor(s):Joyce, Paul
ID Code:983427
Deposited By: MOHAMMED SAMIUR RAHMAN
Deposited On:11 Jun 2018 03:45
Last Modified:11 Jun 2018 03:45

References:

Aebi, M., Kirchner, G., Chen, J. Y., Vijayraghavan, U., Jacobson, A., Martin, N. C., & Abelson, J. (1990). Isolation of a Temperature-Sensitive Mutant With an Altered Transfer-Rna Nucleotidyltransferase and Cloning of the Gene Encoding Transfer-RNA Nucleotidyltransferase in the Yeast (Saccharomyces cerevisiae). Journal of Biological Chemistry, 265(27), 16216–16220.
Arthur, J. (2009). The role of arginine 244 in Candida glabrata tRNA nucleotidyltransferase. Concordia University.
Augustin, M. A., Reichert, A. S., Betat, H., Huber, R., Mörl, M., & Steegborn, C. (2003). Crystal structure of the human CCA-adding enzyme: Insights into template-independent polymerization. Journal of Molecular Biology, 328(5), 985–994.
Berg, J. M., Tymoczko, J. L., Stryer, L. (2010). Biochemistry. W. H. Freeman and Compay, New York.
Betat, H., Rammelt, C., Martin, G., & Mörl, M. (2004). Exchange of regions between bacterial poly(A) polymerase and the CCA-adding enzyme generates altered specificities. Molecular Cell, 15(3), 389–398.
Betat, H., Rammelt, C., & Mörl, M. (2010). TRNA nucleotidyltransferases: Ancient catalysts with an unusual mechanism of polymerization. Cellular and Molecular Life Sciences, 67, 1447–1463.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1–2), 248–254.
Chakraborty, P. K., Schmitz-Abe, K., Kennedy, E. K., Mamady, H., Naas, T., Durie, D., … Fleming, M. D. (2014). Mutations in TRNT1 cause congenital sideroblastic anemia with immunodeficiency, fevers, and developmental delay (SIFD). Blood, 124(18), 2867–2871.
Cho, H. D., Verlinde, C. L. M. J., & Weiner, A. M. (2007). Reengineering CCA-adding enzymes to function as (U,G)- or dCdCdA-adding enzymes or poly(C,A) and poly(U,G) polymerases. Proceedings of the National Academy of Sciences of the United States of America, 104(1), 54–59.
Colasurdo, G. (2011). The role of arginine 204 in Candida glabrata tRNA nucleotidyltransferase. Concordia University.
Creighton, T. E. (1989). Protein structure: a practical approach. IRL. Oxford University Press.
Ernst, F. G. M., Rickert, C., Bluschke, A., Betat, H., Steinhoff; Heinz-Jürgen, & Mörl, M. (2015). Domain movements during CCA-addition : A new function for motif C in the catalytic core of the human tRNA nucleotidyltransferases. RNA Biology, 12(4), 435–446.
Feeney, B., Soderblom, E. J., Goshe, M. B., & Clark, A. C. (2006). Novel protein purification system utilizing an N-terminal fusion protein and a caspase-3 cleavable linker, 47(1), 311–318.
Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel, R. D., & Bairoch, A. (2005). Protein Identification and Analysis Tools on the ExPASy Server. The Proteomics Protocols Handbook, 571–607.
Goring, M. E. (2011). Characterization of temperature-sensitivity and its intragenic suppression in Saccharomyces cerevisiae tRNA nucleotidyltransferase mutants. Concordia University.
Goring, M. E., Leibovitch, M., Gea-Mallorqui, E., Karls, S., Richard, F., Hanic-Joyce, P. J., & Joyce, P. B. M. (2013). The ability of an arginine to tryptophan substitution in Saccharomyces cerevisiae tRNA nucleotidyltransferase to alleviate a temperature-sensitive phenotype suggests a role for motif C in active site organization. Biochimica et Biophysica Acta - Proteins and Proteomics, 1834, 2097–2106.
Hanic-Joyce, P. J., & Joyce, P. B. M. (2002). Characterization of a gene encoding tRNA nucleotidyltransferase from Candida glabrata. Yeast, 19(16), 1399–1411.
Harper, S., Speicher, D. W., & Ph, D. (2011). Protein Chromatography, 681, 1–15.
Hoffmeier, A., Betat, H., Bluschke, A., Günther, R., Junghanns, S., Hofmann, H. J., & Mörl, M. (2010). Unusual evolution of a catalytic core element in CCA-adding enzymes. Nucleic Acids Research, 38(13), 4436–4447.
Kelly, L. A., Mezulis, S., Yates, C., Wass, M., & Sternberg, M. (2015). The Phyre2 web portal for protein modelling, prediction, and analysis. Nature Protocols, 10(6), 845–858.
Klemperer, H. G.; Haynes, G. R. (1967). Altered Specificity of Transfer-Ribonucleic Acid Nucleotidyltransferase in the Presence of Manganese. Biochemistry, 104, 537-544.
Kuhn, C., Wilusz, J. E., Beal, P. a, Joshua-tor, L., Kuhn, C., Wilusz, J. E., … Joshua-tor, L. (2015). On-Enzyme Refolding Permits Small RNA and tRNA Article On-Enzyme Refolding Permits Small RNA and tRNA Surveillance by the CCA-Adding Enzyme. Cell, 160(4), 1–15.
Leatherbarrow, R. J. (2009). GraFit Version 7. Erithacus Software Ltd., Horley, U.K.
Leibovitch, M., Bublak, D., Hanic-joyce, P. J., Tillmann, B., Flinner, N., Amsel, D., … Schleiff, E. (2013). The folding capacity of the mature domain of the dual-targeted plant tRNA nucleotidyltransferase influences organelle selection, 412, 401–412.
Li, F., Xiong, Y., Wang, J., Cho, H. D., Tomita, K., Weiner, A. M., & Steitz, T. A. (2002). Crystal structures of the Bacillus stearothermophilus CCA-adding enzyme and its complexes with ATP or CTP. Cell, 111, 815–824.
Liu, H., & Naismith, J. H. (2008). An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnology, 8(1), 91.
Morris, R. W., & Herbert, E. (1970). Purification and characterization of yeast nucleotidyl transferase and investigation of enzyme-transfer ribonucleic acid complex formation. Biochemistry, 9(24), 4819–27.
Peattie, D. A. (1979). Direct chemical method for sequencing RNA. Proceedings of the National Academy of Sciences of the United States of America, 76(4), 1760–4.
Peltz, S. W., Donahue, J. L., & Jacobson, a. (1992). A mutation in the tRNA nucleotidyltransferase gene promotes stabilization of mRNAs in Saccharomyces cerevisiae. Molecular and Cellular Biology, 12(12), 5778–5784.
Sambrook, J., Maniatis, T., Fritch, E. F. (1989). Molecular Cloning (2nd ed.). Cold Spring Harbor Laboratory Press.
Sasarman, F., Thiffault, I., Weraarpachai, W., Salomon, S., Maftei, C., Gauthier, J., … Shoubridge, E. A. (2015). The 3′ addition of CCA to mitochondrial tRNASer(AGY) is specifically impaired in patients with mutations in the tRNA nucleotidyl transferase TRNT1. Human Molecular Genetics, 24(10), 2841–2847.
Schrödinger. (n.d.). The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC.
Shan, X., Russell, T. A., Paul, S. M., Kushner, D. B., & Joyce, P. B. M. (2008). Characterization of a temperature-sensitive mutation that impairs the function of yeast tRNA nucleotidyltransferase. Yeast, 25, 219–233.
Steitz, T. A. (1998). A mechanism for all polymerases. Nature, 391, 231–232.
Toh, Y., Takeshita, D., Numata, T., Fukai, S., Nureki, O., & Tomita, K. (2009). Mechanism for the definition of elongation and termination by the class II CCA-adding enzyme. The EMBO Journal, 28, 3353–3365.
Tomita, K., Fukai, S., Ishitani, R., Ueda, T., Takeuchi, N., Vassylyev, D. G., & Nureki, O. (2004). Structural basis for template-independent RNA polymerization. Nature, 430, 700–704.
Tomita, K., Ishitani, R., Fukai, S., & Nureki, O. (2006). Complete crystallographic analysis of the dynamics of CCA sequence addition. Nature, 443, 956–960.
Verghese, J., Abrams, J., Wang, Y., & Morano, K. A. (2012). Biology of the Heat Shock Response and Protein Chaperones: Budding Yeast (Saccharomyces cerevisiae) as a Model System. Microbiology and Molecular Biology Reviews, 76(2), 115–158.
Walker, J. M. (2002). Protein handbook (2nd ed.), Humana Press.
Wong, C., Sridhara, S., Bardwell, J. C., Jakob, U. (2000). Heating greatly speeds Coomassie blue staining and destaining. Biotechniques, 28(3), 426–8, 430, 432.
Xiong, Y., Li, F., Wang, J., Weiner, A. M., & Steitz, T. A. (2003). Crystal structures of an archaeal class I CCA-adding enzyme and its nucleotide complexes. Molecular Cell, 12, 1165–1172.
Yakunin, A. F., Proudfoot, M., Kuznetsova, E., Savchenko, A., Brown, G., Arrowsmith, C. H., & Edwards, A. M. (2004). The HD domain of the Escherichia coli tRNA nucleotidyltransferase has 2’,3’-cyclic phosphodiesterase, 2’-nucleotidase, and phosphatase activities. Journal of Biological Chemistry, 279(35), 36819–36827.
Yamashita, S., & Tomita, K. (2016). Mechanism of 3′-Matured tRNA Discrimination from 3′-Immature tRNA by Class-II CCA-Adding Enzyme. Structure, 24(6), 918–925.
Yue, D., Maizels, N., & Weiner, A. M. (1996). CCA-adding enzymes and poly(A) polymerases are all members of the same nucleotidyltransferase superfamily: Characterization of the CCA-adding enzyme from the archaeal hyperthermophile Sulfolobus shibatae. RNA, 2, 895–908.
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