The role of arginine 204 in Candida glabrata tRNA nucleotidyltransferase


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.

PDF - Accepted Version


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.
Date:March 2011
Thesis Supervisor(s):Joyce, Paul
ID Code:7255
Deposited On:13 Jun 2011 09:12
Last Modified:13 Jun 2011 09:12
Arthur, J. (2009) “The role of arginine 244 in Candida glabrata tRNA nucleotidyltransferase” M.Sc. Chemistry thesis, Concordia University.

Augustin, M. A., Reichert, A. S., Betat, H., Huber, R., Morl, M., Steegborn, C. (2003).
Crystal structure of the human CCA-adding enzyme: insights into template-independent
polymerization. J. Mol. Biol. 328, 985-994.

Aravind, L., Koonin, E. V. (1999). DNA polymerase β-like nucleotidyltransferase
superfamily: identification of three new families, classification and evolutionary history.
Nucleic Acids Res. 27, 1609-1618.

Best, A. N., Novelli, G. D., (1971). Studies with adenylyl (cytidylyl) transferase from
Escherichia coli B II. regulation of AMP and CMP incorporation into tRNApCpC and
tRNApC. Arch. Biochem. Biophys. 142, 539-547.

Betat, H., Rammelt, C., Morl, M. (2010). tRNA nucleotidyltransferases: ancient catalysts with an unusual mechanism of polymerization. Cell. Mol. Life Sci. 9, 1447-1463.

Bio-Rad Laboratories. (1995). Sequi-Gen® GT Nucleic Acid Electrophoresis Cell Instruction Manual. Retrieved January 5, 2011, from

Bryson, J. W., Betz, S. F., Lu, H. S., Suich, D. J., Zhou, H. X., O'Neil, K. T., DeGrado, W. F., (1995). Protein design: a hierarchic approach. Science 270, 935-941.

Cho, H. D., Oyelere, A. K., Strobel, S. A., (2003). Use of nucleotide analogs by class I
and class II CCA-adding enzymes (tRNA nucleotidyltransferase): Deciphering the basis
for nucleotide selection. RNA 9, 970-981.

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. Proc. Natl. Acad. Sci. USA 104, 54-59.

Creighton, T. E., (1989). Protein structure: a practical approach. IRL Press at Oxford
University Press, Oxford, p.251-285.

Creighton, T. E., (1993). Proteins: Structures and molecular properties. 2nd ed., W. H.
Freeman and Company, New York, p.171-199 and p.261-328.

Cudny H., Pietrzak, M., Kaczkowski, J., (1978). Plant tRNA nucleotidyltransferase I.
Isolation and purification of tRNA nucleotidyltransferase from Lupinus luteus seeds.
Planta 142, 23-27.

Current Protocols. “DNA/RNA Base Composition Calculator.” Last accessed February 22, 2011.

DeLano, W. L., (2009). PyMOL Molecular Graphics System. DeLano Scientific, LLC.

Deutscher, M. P., (1972a). Reactions at the 3’ terminus of transfer ribonucleic acid. III.
Catalytic properties of two purified rabbit liver transfer ribonucleic acid
nucleotidyltransferases. J. Biol. Chem. 247, 459-468.

Deutscher, M. P., (1973a). Synthesis and Functions of the –C-C-A Terminus of Transfer RNA. Prog. Nucleic Acid Res. Mol. Biol. 13, 51-92.

Deutscher, M. P., (1973b). Reactions at the 3’ terminus of transfer ribonucleic acid. VII.
Anomalous adenosine monophosphate incorporation catalyzed by rabbit liver transfer
ribonucleic acid nucleotidyltransferase. J. Biol. Chem. 248, 3116-3121.

Deutscher, M. P., (1982). tRNA nucleotidyltransferase. In The Enzymes, P. D. Boyer, ed., Academic Press, New York. p.183-215.

Deutscher, M. P., (1983). Enzymes of Nucleic Acid Synthesis and Modification, Volume
II, p168, CRC Press, Boca Raton.

Deutscher, M. P., (1990). Ribonucleases, tRNA nucleotidyltransferase, and the 3’
processing of tRNA. Prog. Nucleic Acid Res. Mol. Biol. 39, 209-240.

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; (In) John M. Walker: The Proteomics Protocols Handbook, Humana Press pp. 571-607.

Gibson, T. J., Higgins, D. G., (2007). ClustalW and ClustalX version 2. Bioinformatics
23, 2947-2948.

Hanic-Joyce, P. J., Joyce, P. B. M., (2002). Characterization of a gene encoding tRNA
nucleotidyltransferase from Candida glabrata. Yeast 19, 1399-1411.

Holm, L., Sander, C., (1995). DNA polymerase β belongs to an ancient nucleotidyltransferase superfamily. Trends Biochem. Sci. 20, 345-347.

Hou, Y. M., (2000). Unusual synthesis by the Escherichia coli CCA-adding enzyme. RNA 6, 1031-1043.

Joyce, C. M., Steitz, T. A., (1994). Function and structure relationships in DNA polymerases. Annu. Rev. Biochem. 63, 777-822.

Just, A., Butter, F., Trenkmann, M., Heitkam, T., Morl, M. Betat, H. (2008). A comparative analysis of two conserved motifs in bacterial poly(A) polymerase and CCA- adding enzyme. Nucleic Acids Res., 36, 5212–5220.

Kelley, L. A., Sternberg, M. J., (2009). Protein structure prediction on the web: a case study using the PHYRE server. MJE Nature Protocols 4, 363-371.

Kim, S., Liu, C., Halkidis, K., Gamper, H. B., Hou, Y. M., (2009). Distinct kinetic determinants for the stepwise CCA addition to tRNA. RNA 15, 1827–1836.

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 and CTP. Cell 111, 815-824.

Lim, K., Ho, J. X., Keeling, K., Gilliland,. G. L., Ji, X., Rüker, F., Carter, D. C., (1994). Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV. Protein Sci. 3, 2233–2244.

Lizano, E., Scheibe, M., Rammelt, C., Betat, H. Morl, M., (2008). A comparative analysis of CCA-adding enzymes from human and E. coli: Differences in CCA addition and tRNA 30-end repair. Biochimie, 90, 762–772.

Martin, G., Keller, W., (1996). Mutational analysis of mammalian poly(A) polymerase
identifies a region for primer binding and a catalytic domain, homologous to the family X
polymerase, and to other nucleotidyltransferases. EMBO J. 15, 2593-2603.

McGann, R. G., Deutscher, M. P. (1980). Purification and Characterization of a Mutant tRNA Nucleotidyltransferase Eur. J. Biochem. 106, 321–328.

Milligan, J. F. & Uhlenbeck, O. C. (1989). Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymology 180, 51-62.

Neuenfeldt, A., Just, A., Betat, H., Morl, M., (2008). Evolution of tRNA nucleotidyltransferases: a small deletion generated CC-adding enzymes. Proc. Natl. Acad. Sci. USA, 105, 7953–7958.

Oh, B., Pace, N. R., (1994). Interaction of the 3’-end of tRNA with ribonuclease P
RNA. Nucleic Acids Res. 22, 4087-4094.

Pain, R. H., (2004). Determining the Fluorescence Spectrum of a Protein. Curr. Protoc. Protein Sci. 38, 7.7.1-7.7.20.

Peattie, D.A., (1979). Direct chemical method for sequencing RNA. Proc. Natl. Acad. Sci. USA 76, 1760-1764.

Pleiss, J. A., Derrick, M. L., Uhlenbeck, O. C., (1998). T7 RNA polymerase produces 5’-end heterogeneity during in vitro transcription from certain templates. RNA 4, 1313–1317.

Rether, B., Gangloff, J., Ebel, J. P., (1974). Studies on tRNA nucleotidyltransferase
from baker’s yeast 2. Replacement of the terminal CCA sequence in Yeast tRNAPhe
by several unusual sequences. Eur. J. Biochem. 50, 289-295.

Sambrook, J., Maniatis, T., Fritch, E.F., (1989). Molecular Cloning 2nd Ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor.

Schürer, H., Lang, K., Schuster, J., Morl, M., (2002). A universal method to produce in vitro transcripts with homogenous 3´ends. Nucleic Acids Res 30, e56.

Seth, M., Thurow, D. L., Hou, Y-M., (2002). Poly(C) synthesis by Class I and Class II
CCA-adding enzymes. Biochemistry 41, 4521-4532.

Shan, X., (2005). Characterization of a temperature-sensitive mutation that impairs the function of yeast tRNA nucleotidyltransferase. M.Sc. Chemistry thesis, Concordia University, Montreal.

Shi, P. Y., Weiner, A. M., Maizels, N., (1998a). A top-half tDNA minihelix is a good substrate for the eubacterial CCA-adding enzyme. RNA, 4, 276–284.

Shi, P. Y., Maizels, N. and Weiner, A. M., (1998b). CCA addition by tRNA nucleotidyltransferase: polymerization without translocation. EMBO J. 17, 3197–3206.

Sikorski R.S., Hieter, P., (1989). A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19-27.

Steitz, T. A., (1998). A mechanism for all polymerases. Nature 391, 231–232.

Steitz, T. A., Smerdon, S. J., Jager, J., Joyce, C. M., (1994). A unified polymerase mechanism for nonhomologous DNA and RNA polymerases. Science 266, 2022–2025.

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. EMBO J. 28, 3353-65.

Tomita, K. and Weiner, A. M., (2001). Collaboration between CC- and A-adding enzymes to build and repair the 3’-terminal CCA of tRNA in Aquifex aeolicus. Science 294, 1334-1336.

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.

Walker, J. M., (2002). The Protein Protocols Handbook. Humana Press, p. 57-67.

Wong, C., Sridhara, S., Bardwell, J. C. A., Jakob, U., (2000). Heating greatly increases
speed of Coomassie Blue staining and destaining. Biotechniques 28, 426-432.

Yamada, Y. M., Ohki, H., Ishikura, H., (1983). The Nucleotide sequence of Bacillus subtilis tRNA genes. Nucleic Acids Res. 11, 3037-3045.

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.

Yue, D., Weiner, A. M., Maizels, N., (1998). The CCA-adding enzyme has a single active site. J. Biol. Chem. 273, 29693-29700.

Zhu, L., Cudny, H. Deutscher, M. P., (1986). Mutation in Escherichia coli tRNA nucleotidyltransferase that affects only AMP incorporation is in a sequence often associated with nucleotide-binding proteins. J. Biol. Chem. 261, 14875-14877.
All items in Spectrum are protected by copyright, with all rights reserved. The use of items is governed by Spectrum's terms of access.

Repository Staff Only: item control page

Document Downloads

More statistics for this item...

Concordia University - Footer