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Kimberly Durniak Center for Structural Biology Phone:
(203) 432-5625 Email: kimberly.durniak {at} yale.edu |
Using crystallographic techniques to further understand bacteriophage T7 RNA Polymerase mechanism
Summary:
Polymerase is the key performer in Crick’s Central Dogma of molecular biology: DNA à RNA à Protein. DNA-dependent RNA polymerases are essential for their specificity in transcribing DNA into RNA. The processive RNA polymerase (RNAP) from the bacteriophage T7 is part of a transcription system used widely to obtain recombinant proteins. T7 RNAP provides a good model for understanding transcription because of its small size, 98kDa, relative to large multisubunit eukaryotic polymerases and the large amount of supporting biochemical and structural data. Transcription by T7 RNAP, like the larger polymerases, has four distinct phases: initiation, abortive cycling, elongation and termination. Transcription of T7 RNAP can be tightly controlled by T7 lysozyme, a process that is inherent to manipulating this system (1).
The first crystal structure from this system, of apo T7 RNAP solved to 3.3Å, revealed that the fingers, palm and thumb subdomains seen in the Klenow fragment are also present in T7 RNAP (2). The initiation complex solved by Cheetham and Steitz contains DNA and a RNA nascent chain three nucleotides in length (3). From this structure it was clear that the protein had to undergo some structural rearrangement in order to incorporate more than 3 nucleotides due to the steric clashes with the protein main-chain. The structure of the elongation complex was solved by two different groups, Yin from the Steitz group, and Tahirov from the Yokoyama group (4, 5). The elongation complex structure clarified much of the biochemical evidence supporting a structural change in the protein during the transition from the initiation phase to the elongation phase(figure 1). One aim of this project is to explore the structural changes that occur during the transition from initiation to elongation in order to understand the trigger for the refolding event allowing for processivity.
Biochemical work has shown through transcription assays that T7 lysozyme inhibits the transition to elongation. Lysozyme binds both the initiation and the elongation complexes (1, 6), and interestingly, the T7 RNAP:lysozyme structure(7), lacking nucleic acids, is also bound by lysozyme. A comparison of the structure of T7 RNAP:lysozyme complex with the structure of the elongation complex reveals that the region of lysozyme binding does not undergo a structural change during the transition from initiation to elongation. This poses the question of how lysozyme inhibits a transition when its physical interaction with the polymerase does not seem to change at the start and end points of this transition, i.e. initiation and elongation. Could lysozyme interfere with the forming of a transition complex? Or are its effects unrelated to the polymerase refolding? I aim to answer these questions by seeking crystal structures of T7 RNAP: Lysozyme complexes bound to DNA/RNA substrates that mimic initiation through to elongation.
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Publication:
Commentary on this work:
1. B. A. Moffatt, F. W. Studier, Cell 49, 221-227 (1987).
2. R. Sousa, Y. J. Chung, J. P. Rose, B.-C. Wang, Nature 364, 593-599 (12 August 1993).
3. G. M. T. Cheetham, T. A. Steitz, Science 286, 2305-2309 (1999).
4. T. H. Tahirov et al., Nature 420, 43-50 (7 November 2002).
5. Y. W. Yin, T. A. Steitz, Science 298, 1387-1395 (15 November 2002).
6. J. Huang, J. Villemain, R. Radilla, R. Sousa, Journal of Molecular Biology 293, 457-475 (1999).
7. D. Jeruzalmi, T. A. Steitz, EMBO 14, 4101-4113 (1998).