Faculty and Research Interests

William T. McAllister, PhD

Professor and Chair
Department of Cell Biology
Science Center 215a
2 Medical Center Drive
Stratford, NJ 08084
mcalliwt@umdnj.edu

Work in our laboratory is directed at understanding fundamental aspects of the transcription process, and in particular, how the enzyme that carries out this process works. As a model system we have chosen the relatively simple RNA polymerase (RNAP) that is encoded by bacteriophage T7.

Figure 1. Structure of the "DNA zipper" which separates the template (red) and non-template (blue) DNA strands at the leading edge of the transcription bubble. RNA is yellow.

Although this enzyme consists of a single subunit it carries out all of the steps in the transcription process as the multisubunit RNAPs found in prokaryotic and eukaryotic cells. Due to its structural simplicity and ease of genetic manipulation the phage enzyme provides particular advantages for studies of RNAP structure and function. In our work we use biochemical and genetic methods to characterize the importance of various regions of the RNAP to RNA synthesis, and interpret the results with regard to the organization of the enzyme as determined by crystallographic analysis and other methods of structural determination.

A number of structures of T7 RNAP have been solved, including: free RNAP, RNAP bound to the promoter, an early initiation complex, and, most recently, the structure of an elongation complex. These structures, together with structures of bacterial and yeast RNAPs, have provided a wealth of information concerning common features of the transcription machinery, and important insights into the transcription process. Nevertheless, important gaps remain in our knowledge of the various stages of transcription, most importantly with regard to the transition that leads from an unstable initiation complex (IC) to a stable elongation complex (EC), and with regard to the process of termination.

Figure 2. Summary of protein: nucleic acid interactions in a T7 RNA polymerase elongation complex. Circles indicate bases in the template (white) or non-template (blue) strands of the DNA, or in the RNA product (red), with regions of the RNA polymerase that include the amino acid residues indicated.

To characterize T7 RNAP function we isolate or construct mutant enzymes with defined biochemical defects, and ask whether these defects can be correlated with the structure of the enzyme as determined by X-ray crystallography. These studies have led to the characterization of a number of important functional domains in the enzyme; for example, domains that are involved in promoter recognition, RNA displacement, stabilization of the elongation complex, and termination.

To examine the organization of transcription complexes, we use nucleic acid:protein crosslinking methods to determine the trajectory of the DNA and the RNA over the surface of the enzyme. Mapping the sites of these crosslinks onto the three dimensional structure of the RNAP has provided important insights into the organization of the complex, and of the events that lead from an IC to an EC. Recently, in collaboration with Drs. Vassylyev, Tahirov, and Yokoyama at the RIKEN Harima Institute, Hyogo, Japan, we have begun to analyze the structure of additional T7 transcription complexes using X-ray crystallographic methods.

Figure 3. Details of RNA product displacement. Electrostatic and steric interactions at the upstream edge of the RNA:DNA hybrid separate the RNA from the DNA template, directing the RNA towards a positively charged exit pore.

In addition to their utility in studies of the transcription process, phage RNAPs are critical to a number of practical applications, including high level expression systems and the synthesis of nucleic acid probes. Many of the mutant enzymes that we have characterized have altered properties that are useful in these applications, and we are working to develop and improve these technologies. Lastly, we have initiated experiments to explore the use of RNA polymerases as an information-dependent molecular motor; we believe that these studies have important implications in nanotechnology and information sciences

Recent Publications

1. Kukarin, A., Rong, M.R., McAllister, W.T. (2003). Exposure of T7 RNA polymerase to the double stranded binding region of the promoter activates the enzyme to transcribe a single stranded template, J. Biol. Chem. 278:2419-2424

2. Temiakov,D.; Tahirov,T.; Anikin,M.; McAllister,W.T.; Vassylyev,D.G.; Yokoyama,S. (2003). Crystallization and preliminary crystallographic analysis of T7 RNApolymerase elongation complex assembled on an RNA:DNA scaffold. Acta Crystallographica 59:185-187

3. Tahirov,T.; Temiakov,D.; Anikin,M.; Patlan,V.; McAllister,W.T.; Vassylyev,D.G.; Yokoyama,S. (2002). Structure of a T7 RNA polymerase elongation complex at 2.9Å resolution. Nature 420:43-50.

4. Temiakov,D.; Anikin,M.; McAllister,W.T. (2002). Characterization of T7 RNA polymerase transcription complexes assembled on nucleic acid scaffolds. J. Biol. Chem. 277:47035-47043.

5. Ma,K.; Temiakov,D.; Jiang,M.; Anikin,M.; McAllister,W.T. (2002). Major conformational changes occur during the transition from an initiation complex to an elongation complex by T7 RNA polymerase. J. Biol. Chem. 277:43206-43215.

6. Imburgio, D. Anikin, M., McAllister, W.T. (2002). Effects of substitutions in a conserved DX2GR motif found in many DNA-dependent nucleotide polymerases on transcription by T7 RNA polymerase. J. Mol. Biol. 319: 37-51

7. Jiang M, Rong, M, Martin CT, McAllister WT. (2001). Interrupting the template strand of the T7 promoter facilitates translocation of the DNA during initiation, reducing transcript slippage and the release of abortive products. J. Mol. Biol. 310: 509-522.

8. Temiakov, D., Mentesana, P.E., Ma, K., Mustaev, A., Borukhov, S. McAllister, W.T. (2001). The specificity loop of T7 RNA polymerase interacts first with the promoter and then with the elongating transcript, suggesting a mechanism for promoter clearance. Proc. Nat. Acad. Sci. (USA), 97: 14109-14114. See Commentary by K. Severinov, Proc. Nat. Acad. Sci. (USA), 98: 5-7

9. Mentesana,P.E., Chin-Bow, S.T., Sousa, R., and McAllister, W.T. (2000). Characterization of halted T7 RNA polymerase elongation complexes reveals multiple factors that contribute to stability. J. Mol. Biol. 302:1049-1062.

10. Imburgio D, Rong M, Ma K, McAllister WT. (2000). Studies of promoter recognition and start site selection by T7 RNA polymerase using a comprehensive collection of promoter variants. Biochemistry 39: 10419-10430.

11. Rong M, Castagna RC, McAllister WT. (1999). Cloning and purification of bacteriophage K11 RNA polymerase. Biotechniques 27, 692-693.

12. Place C, Oddos J, Buc H, McAllister WT, Buckle M. (1999). Studies of contacts between T7 RNA polymerase and its promoter reveal features in common with multisubunit RNA polymerases. Biochemistry 38:4948-4957

13. Gopal V, Brieba LG, Guajardo R, McAllister WT, Sousa R. (1999). Characterization of structural features important for T7 RNAP elongation complex stability reveals competing complex conformations and a role for the non-template strand in RNA displacement. J. Mol. Biol. 290,411-431.

14. He, B., Kukarin, A., Temiakov, D., Chin-Bow, S.T., Lyakhov, D.L., Rong, M., Durbin, R.K. & McAllister, W.T. (1998). Characterization of an unusual, sequence-specific termination signal for T7 RNA polymerase. J. Biol. Chem. 273,18802-18811.

15. Lyakhov, D.L., He, B., Zhang, X., Studier, F.W., Dunn, J.J. & McAllister, W.T. (1998). Pausing and termination by bacteriophage T7 RNA polymerase. J.Mol.Biol. 280: 201-213.

16. Rong, M., Durbin, R.K. & McAllister, W.T. (1998). Template strand switching by T7 RNA polymerase. J.Biol.Chem. 273: 10253-10260.

17. Rong, M., He, B., McAllister, W.T. & Durbin, R.K. (1998). Promoter specificity determinants of T7 RNA polymerase. Proc.Nat.Acad.Sci.U.S.A. 95: 515-519.