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Andrew Bohm, Ph.D. Associate Professor, Tufts Department of Biochemistry Adjuct Scientist, Boston Biomedical
Research Institute B.S. Biochemistry, State University of New York at Binghamton Ph.D. Biophysics, University of California at Berkeley |
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Links:Research
Lab MembersRecent Publications |
Research Summary:We use X-ray crystallography in conjunction with other biochemical and biophysical techniques to study the structure and function of the roughly one dozen proteins which together constitute the yeast cleavage/polyadenylation complex. Similar macromolecular assemblies are present in all eukaryotic organisms, and serve to truncate and polyadenylate the 3' end of messenger RNA prior to nuclear export. We are also working along with Dr. Peter Bullock in the Biochemistry Department to understand how SV40 T-antigen and the large T-antigen molecules from other viruses interact with DNA during origin recognition and the initiation of DNA replication. |
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Polyadenylation is an essential step in mRNA synthesis, and defects in this process profoundly interfere with normal cell function. In humans such defects have been shown to cause thalassemias, lysozomal storage disorders, and a form of muscular dystrophy. The poly(A) polymerase (PAP) responsible for post-transcriptional mRNA elongation in eukaryotes does not require a template strand. As such, this enzyme is fundamentally different from the vast majority of other polymerases that have been structurally characterized. To form a poly(A) tail, PAP has evolved a mechanism that allows its amino acids to encode specificity for RNA as a primer and adenosine as the base of the incoming nucleotide triphosphate.
Our crystal structure of Poly(A)
polymerase (PAP) shows that the enzyme is organized into three domains
of 150-200 amino acids each. The N-terminal domain belongs to the
nucleotidyl transferase (NT) superfamily of enzymes, and contains the
hallmark triad of carboxylic amino acids. These carboxylic amino acids
participate in the metal-coordinated binding of the ATP phosphate
groups, and are responsible for positioning the nucleophilic 3'-OH of
the primer to facilitate the nucleotidyl transferase reaction.
Mutagenesis studies have implicated residues within the middle domain
in differentiating between RNA and DNA primers, and in binding a
variety of factors which regulate polyadenylation. The C-terminal
region of PAP is responsible for holding the single stranded poly(A)
primer in place. Template-directed polymerases use a "thumb domain" to
hold onto their substrate via the template strand. Since PAP does not
utilize a template, the thumb domain is missing, and the responsibility
for holding the substrate has been largely shifted to the C-terminal
domain. In our crystal structure, we see two molecules of 3'deoxy-ATP
bound to the enzyme. One (shown in red) binds the expected position of
the incoming nucleotide. The other (yellow) binds a site that is
biochemically consistent with that expected for the 3' end of the mRNA
substrate. Curiously, we do not see any clear interactions between the
incoming nucleotide and the base of the incoming ATP, suggesting that
the protein and/or nucleotide must adopt some other conformation during
the part of the reaction in which the base is recognized by the enzyme.
Our current research in this area is focused on understanding the structural basis for PAP processivity. In addition to PAP itself, we are also pursuing crystallization trials involving a variety of proteins which regulate PAP. In the cell, PAP exists as part of the multi-component cleavage/polyadenylation macromolecular assembly. Components of this assembly (most notably Fip1 in the yeast system) regulate both activity and processivity.
Meinke, G., Ezeokonkwo, C., Balbo, P., Stafford, W., Moore, C., and Bohm, A. Structure of yeast poly(A) polymerase in complex with a peptide from Fip1, an intrinsically-disordered protein. (2008) (in press, Biochemistry).
Lucia Morstadt, Andrew Bohm, Deniz Yüksel, Krishna Kumar, B. David Stollar, and James D. Baleja. (2008) Engineering and characterization of a single chain surrogate light chain variable domain. Protein Sci. 17: 458-465.
Balbo P and Bohm A. (2007) Mechanism of Poly(A) Polymerase: Structure of the enzyme-MgATP-RNA ternary complex and kinetic analysis. Structure. 15(9):1117-31.
Kumar A, Meinke G, Reese DK, Moine S, Phelan PJ, Fradet-Turcotte A, Archambault J, Bohm A, Bullock PA. (2007) Model for T-antigen-dependent melting of the simian virus 40 core origin based on studies of the interaction of the beta-hairpin with DNA. J Virol. 81(9):4808-18.
Balbo, P., Toth, J., Meinke, G., Bohm, A. (2007) X-ray crystallographic and steady state fluorescence characterization of the protein dynamics of yeast polyadenylate polymerase. Journal of Molecular Biology. 366(5):1401-15.
Meinke, G., Phelan, P., Moine, S., Bochkareva, E., Bochkarev, A., Bohm, A. (2007) The Crystal Structure of the SV40 T-Antigen Origin Binding Domain in Complex with DNA. PloSBiology: e23.
Reese, DK, Meinke, G, Kumar, A, Moine, S, Chen, K, Sudmeier, JL, Bachovchin, W, Bohm, A, Bullock, PA. (2006) Analysis of the Transit of DNA through T-antigen Hexamers ProvidesInsights into a Eukaryotic Helicase and the Initiation of Simian Virus 40 DNA Replication. J Virology. 80(24):12248-59.
Swift, Steven; Leger, Andrew J; Talavera, Joyce; Zhang, Lei; Bohm, Andrew; Kuliopulos, Athan. (2006). “Role of the PAR1 Receptor 8th Helix in Signaling: The 7-8-1 Receptor Activation Mechanism.” Journal of Biological Chemistry 281(7): 4109-4116.
Meinke, Gretchen; Bullock, Peter A; Bohm, Andrew. (2006). “Crystal structure of the SV40 large T-antigen origin-binding domain.” Journal of Virology 80(9): 4304-4312.
Balbo, PB., Meinke, G. and Bohm, A. (2005) Kinetic studies of yeast poly(A) polymerase indicate an induced fit mechanism for nucleotide specificity. Biochemistry. 44(21): 7777-86.
Zhelkovsky, A, Helmling, S., Bohm, A., Moore, C. (2004) Mutations in the middle domain of yeast poly(A) polymerase affect interactions with RNA but not ATP. RNA 10(4): 558-64.
Shen, Y., Guo, Q., Zhukovskaya, N.L., Drum, C.L., Bohm, A., Tang, W.-J. Structure of anthrax edema factor-calmodulin sensitive adenosine-(alpha, beta-methylene)-triphosphate complex reveals an alternative mode of ATP binding to the catalytic site. (2004) Biochem Biophys Res Commun. 317(2) 309-14.
Obin, M., Leyy, B.Y., Meinke, G., Bohm, A., Lee, R.H., Gaudet, R., Hopp, J.A., Arshavsky, V.A., Willardson, B.A., Taylor, A. (2002) Ubiquination of the Trasducin beta gamma Subunit Complex. Journal of Biological Chemistry 277, 44566-44575.
Drum, C.L., Yan, S.-Z., Bard, J., Shen, Y.-Q., Lu, D., Soelaiman, S., Grabarek, Z., Bohm, A., Tang, W.-J. (2002) Structural basis for the activation of the anthrax adenylyl cyclase exotoxin by calmodulin. Nature 415, 396-402.
Drum, C.L., Shen, Y., Rice, P.A., Bohm, A., Tang, W.-T. Crystallization and Preliminary X-ray Study of the Edema Factor Exotoxin Adenylyl Cyclase Domain from Bacillus Anthracis in the Presence of its activator, Calmodulin. (2001) Acta Crystallographica D57, 1881-1884.
Skiba N.P., Martemyanov, K.A., Elfeinbein, A., Hopp, J.A., Bohm, A., Simonds, W.F., and Arshavsky, V.Y. (2001) GS9-G beta 5 substrate selectivity in photoreceptors. Opposing effects of constituent domains yield high affinity of RGS interaction with the G protein-effector complex. Journal of Biological Chemistry 276:37365-72.
Drum, C.L., Yan, S.-Z., Sarac, R., Mabuchi, Y., Beckingham, K., Bohm, A., Grabarek, Z., Tang, W.-J. (2000) An Extended Conformation of Calmodulin Induces Interactions between the Structural Domains of Adenylyl Cyclase from Bacillus anthracis to Promote Catalysis. Journal of Biological Chemistry 275, 36334-340.
Bard J, Zhelkovsky AM, Helmling S, Earnest TN, Moore CL, Bohm A. (2000) Structure of yeast Poly(A) polymerase alone and in complex with 3'-dATP. Science 289, 1346-1349.
| Gretcheb Meinke, Ph.D. (Research
Associate) Gretchen.Meinke@tufts.edu |
| Sergei Shikov, Ph.D. (Postdoctoral
Associate) Sergei.Shikov@tufts.edu |
| James Gordon, Ph.D. (Postdoctoral
Associate) James.Gordon@tufts.edu |
