Cell Biology
Faculty and Research Interests
William T. McAllister, PhD
Professor and Chair
Science Center 215a
mcalliwt@umdnj.edu
Education
Department of Biochemistry, University of New Hampshire, NH
PhD , 1970
Research Interests
Figure 1. Model of an EC with a misaligned Base Comparison of the structure of the T7 RNAP EC observed in the open configuration in the presence of the correct incoming NTP (left) and a model of a misaligned EC in which the TSn base is rotated out of the preinsertion site, allowing the TSn+1 base to pair with the incoming substrate (right). The protein is shown as a transparent surface; DNA template strand (TS) is red, RNA primer yellow, incoming NTP blue, TSn base white, TSn+1 base light red, and Y and Z helices (residues 624 to 658) and side chains of residues implicated in fidelity (Y639, G640, F644, G645, and H784) are in pink (See Pomerantz et al, Mol. Cell, 2006).
Work in our laboratory is directed at understanding fundamental aspects of the transcription process. As a model system we have chosen the relatively simple RNA polymerase (RNAP) that is encoded by bacteriophage T7. Although this enzyme consists of a single subunit, it carries out all of the steps in the transcription process in a similar manner as the multisubunit RNAPs found in eukaryotic cells. Importantly, the phage enzyme is structurally related to other nucleotide polymerases of the pol I family, such as DNA polymerase I and reverse transcriptase. It therefore affords an opportunity to explore and compare general features of nucleotide polymerization and fidelity among members of this family, as well as with multisubunit enzymes.
In our work we use biochemical and genetic methods to characterize the functional importance of various regions of the RNAP, 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. 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. (See Jiang et al, Mol Cell. 2004)
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. In addition, 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.
Lastly, we note that mitochondria, which are responsible for production of most energy in eukaryotic cells and are thought to be derived from an ancient eubacterial endosymbiont, contain a DNA genome that is transcribed by an RNAP that is structurally related to T7 RNAP. Despite the importance of mitochondrial function, relatively little is known about the mitochondrial transcription apparatus, its regulation, or what proteins may be associated with the transcription complex during different stages of development, disease, and/or oxidative stress. Based upon our knowledge of T7 RNAP, we have begun to characterize the mitochondrial transcription system in some detail.
Publications
- 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.
- 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.
- Temiakov, D., Patlan,V., Anikin, M., McAllister, W.T., Yokoyama, S., Vassylyev, D.G. (2004), Structural basis for substrate selection by T7 RNA polymerase, Cell 116: 381-391. See commentary by R. Landick, Cell 116: 351-353.
- Jiang, M., Ma,N., Vassylyev, D.G., McAllister, W.T. (2004), RNA displacement and resolution of the transcription bubble during transcription by T7 RNA polymerase, Mol. Cell 15:777-788.
- Pomerantz, R.T., Ramjit, R. Gueroui, Z., Place, C., Anikin, M., Leuba, S., Zlatanova, J., McAllister, W.T. (2005), A tightly regulated molecular motor based upon T7 RNA polymerase. Nano Lett.5:1698-1703.
- Ma, K., Temiakov, D., Anikin, M., McAllister, W.T. (2005), Probing conformational changes in T7 RNA polymerase during initiation and termination using engineered disulfide linkages, Proc. Nat. Acad. Sci (USA) 102:17612-17617.
- Zlatanova, J., McAllister, W.T., Borukhov, S. Leuba, S.H. (2006), Single-Molecule Approaches Reveal the Idiosyncrasies of RNA Polymerases. Structure 14:953-966.
- Pomerantz, R.T., Temiakov, D., Anikin, M, Vassylev, D.G., McAllister, W.T. (2006), A novel mechanism of nucleotide misincorporation during transcription due to template strand misalignment, Mol. Cell 24:245–255.
- Kashkina, E., Anikin, M, Brückner, F., Pomerantz, R.T., McAllister, W.T., Cramer, P., Temiakov, D. (2006), Template misalignment in multisubunit RNA polymerases and transcription fidelity. Mol. Cell 24:257-256.
- Kashkina, E., Anikin, M., Brueckner, F., Lehmann, E., Kochetkov, S.N., McAllister, W.T., Cramer, P., Temiakov, T. (2007), Multi-subunit RNA polymerases melt only a single DNA base pair downstream of the active site. J. Biol. Chem. 30:2158-2182.
- Bandwar, R.P., Ma, N., Emanuel, S.A., Anikin, M., Vassylyev, D.G., Patel, S.S., McAllister, W.T. (2007), The transition to an elongation complex by T7 RNA polymerase is a multistep process. J. Biol. Chem. 31:22879-22886.
