Progress 10/01/05 to 09/30/13
Outputs
OUTPUTS: Our outputs for the last year include new results on the mechanism and regulation of transcript elongation by RNA polymerase. We have reported the novel finding that the pause and termination activity of NusA resides in its N-terminal domain. Additionally, based on in vitro assays of Escherichia coli RNA polymerase bearing specific alterations in the trigger loop, we found that neither intrinsic nor regulator-assisted transcript cleavage of backtracked RNA requires formation of the trigger helices. We also found that the principal contribution of the trigger helices to rapid nucleotidyl transfer is steric alignment of the reactants, rather than acid-base catalysis, and that the trigger loop cannot be the sole contributor to substrate selectivity. The similar effects of trigger loop substitutions on pausing and nucleotide addition provided additional support for the view that trigger helices formation is rate-limiting for escape from nonbacktracked pauses. Additionally, in collaboration with S. Darst at Rockefeller U. we reported a structural model of E. coli RNA polymerase. All of these studies have generated new knowlege that represent our principle outputs. We have disseminated these findings in the past year both through presentations at meetings and by publications in the scientitifc literature. These include presentations at the 2010 Transcription Termination meeting in Mt. Lake, VA and the 2010 Biochemical Society meeting on RNA Polymerase in Cambridge,UK. Additionally, seminars were given at the University of Alabama-Birmingham and Publications are reported below. PARTICIPANTS: The following individuals worked on the project. Rachel Mooney, Murali Palangat, Frances Tran, Jeff Grass; Postdocs: Agata Czyz, Dhananjaya Nayak, Rembrandt Haft, Matt Kotlajich; Graduate Students: Kook Sun Ha, Abbey Vangeloff, Pyae Hein, Jason Peters, Mike Frazier; Thus we provided training opportunities and professional development for 4 potdocs and 5 graduate students. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our work led to important new results and conclusions about RNA polymerase and the regulation of transcript elongation. Our studies on NusA redefined understanding of its structure/function relationship. Our findings on the trigger loop provided key insight into this critical component of the catalytic center of this important enzyme. Finally, we found that sequence insertions in the E. coli RNA polymerase allow it to evolve more efficiently to growth on new media.
Publications
- Zhang, J., M. Palangat, and R. Landick. 2010. Role of the RNA polymerase trigger loop in nucleotidyl transfer, transcript cleavage, and transcriptional pausing. Nat. Struct. Mol. Biol., 17, 99-104.
- Herbert, K.M., Zhou, J., Mooney, R.A., Porta, A.L., Landick, R., and Block, S.M. 2010. E. coli NusG inhibits backtracking and accelerates pause-free transcription by promoting forward translocation of RNA polymerase. J Mol Biol., 399, 17-30.
- Ha, K. S., Toulokhonov, I., Vassylyev, D. G. & Landick, R. 2010. The NusA N-terminal domain is necessary and sufficient for enhancement of transcriptional pausing via interaction with the RNA exit channel of RNA polymerase. J Mol Biol 401, 708-25. PMCid in process
- Cohen, S. E., Lewis, C. A., Mooney, R. A., Kohanski, M. A., Collins, J. J., Landick, R. & Walker, G. C. 2010. Roles for the transcription elongation factor NusA in both DNA repair and damage tolerance pathways in Escherichia coli. Proc Natl Acad Sci U S A 107, 15517-22.
- Huff, J., Czyz, A., Landick, R. & Niederweis, M. 2010. Taking phage integration to the next level as a genetic tool for mycobacteria. Gene 468, 8-19.
- Opalka, N., Brown, J., Lane, W. J., Twist, K. A., Landick, R., Asturias, F. J. & Darst, S. A. (2010). Complete structural model of Escherichia coli RNA polymerase from a hybrid approach. PLoS Biol 8, e1000483.
- Conrad, T., M. Frazier, A. Joyce, B. Cho, E. Knight, N. Lewis, R. Landick,* and B. Palsson. 2010. RNA polymerase mutants found through adaptive evolution re-program Escherichia coli for optimal growth in minimal media. Proc. Natl. Acad. Sci. U. S. A. 107, 20500-20505.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Our outputs for the last year include new results on the mechanism and regulation of transcript elongation by RNA polymerase. We have studied the effects of inhibiting the elongation factor Rho on the distribution of RNA polymerase on bacterial transcription units. We also have studied the influence of DNA sequence on the translocation register of RNA polymerase. Finally, we have studied the mechanisms of action of elongation regulators NusA and NusG. All of these studies have generated new knowlege that represent our principle outputs. We have disseminated these findings in the past year both through presentations at meetings and by publications in the scientitifc literature. These include presentations at the 2008 Molecular Genetics of Bacteria meeting in Madison, WI, the 2008 Eukaryotic Transcription Meeting at Cold Spring Harbor, NY, the American Society of Biochemistry and Molecular Biology meeting in New Orleans, the American Society for Microbiology meeting in Philadelphia, and the 2008 FASEB meeting on Prokaryotic Gene Regulation in Saxtons River,VT. Publications are reported below. PARTICIPANTS: The following individuals worked on the project. Rachel Mooney, Murali Palangat, Frances Tran, Jeff Grass; Postdocs: Agata Czyz, Dhananjaya Nayak, Rembrandt Haft; Graduate Students: Kook Sun Ha, Abbey Vangeloff, Pyae Hein, Jason Peters, Jinwei Zhang, Mike Frazier; Thus we provided training opportunities and professional development for 3 potdocs and 6 graduate students. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our work over the past year has led to important new results and conclusions about RNA polymerase and the regulation of transcript elongation. Our studies on the effects of inhibition of Rho led to discovery of a new class of antisense transcripts in the bacterial genome. We also established that the translocation register of RNA polymerase was controlled principally by the sequence of the DNA:RNA hybrid in the active site of the enzyme. Finally, we found that a key part of the RNA polymerase called the trigger loop, which plays critical roles in the transcript elongation, was not required for either transcript cleavage or transcription termination.
Publications
- Belogurov G. A., R. A. Mooney, V. Svetlov, R. Landick I. Artsimovitch. 2009. Functional specialization of transcription elongation factors. EMBO J. 28, 112-122
- Landick, R. 2009. Functional divergence in the growing family of RNA polymerases. Structure, 17, 323-325
- 33. Landick, R. 2009. Transcriptional pausing without backtracking. Proc. Natl. Acad. Sci. U.S.A., 106, 8797-8798.
- Mooney, R.A., S.E. Davis, J. M. Peters, J. L. Rowland, A. Z. Ansari, and R. Landick. 2009. Regulator Trafficking on bacterial transcription units. Mol. Cell., 33, 97-108.
- Mooney, R.A., K. Schweimer, P. Rosch, M. Gottesman and R. Landick. 2009. Two structurally independent domains of E. coli NusG create regulatory plasticity via distinct interactions with RNA polymerase and regulators. J. Mol. Biol. 391, 341-358.
- Peters, J.M., R.A. Mooney, P.F. Kuan, J. L. Rowland, S. Keles, and R. Landick. 2009. Rho directs widespread termination of intragenic and stable RNA transcription. Proc. Natl. Acad. Sci. U.S.A. 106, 15406-15411.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: New insights were gained into the function of RNA polymerase in bacteria and human cells. This knowledge improves understanding of how gene expression is regulated. The following specific insights were gained. 1. The RNA polymerase trigger loop is required for nucleotidyl transferase activity but not for intrinsic cleavage activity of RNA polymerase. 2. The NusA transcription regulator contains all of its pause- and termination-stimulating activity in its N-terminal domain. 3. The NusG transcription regulator binds to elongation complexes in competition with the sigma initiation factor. 4. The Rho transcription regulator binds to all transcription units in vivo, even though it is active at a subset of sites. 5. Contrary to earlier views, the Rho transcription regulator terminates transcription of small RNAs. The new understanding was disseminated to other scientists at research meetings, in addition to publications. PARTICIPANTS: Robert Landick, Rachel Mooney, Murali Palangat, Agata Czyz, Jason Peters, Jinei Zhang, Kook Sun Ha, Abbey Vangeloff, Pyae Hein, Mike Frazier TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Experimental research was conducted to produce the findings. This research used a variety of methods of biochemistry and molecular biology, including in vitro transcription, protein purification, in vivo ChIP chip assays, protein footprinting, and real-time PCR.
Publications
- Westblade, L.F., Minakhin, L., Kuznedelov, K., Tackett, A.J., Chang, E.J., Mooney, R.A., Vvedenskaya, I., Wang, Q.J., Fenyo, D., Rout, M.P., Landick, R,. Chait, B.T., Severinov, K., Darst, S,A. 2008. Rapid isolation and identification of bacteriophage T4-encoded modifications of Escherichia coli RNA polymerase: a generic method to study bacteriophage/host interactions. J. Proteome Res., 7, 1244-1250.
- Dufour, Y., Landick, R., and Donohue, T. 2008. Organization and evolution of the biological response to singlet oxygen stress. J. Mol. Biol., 383, 713-730.
- Larson, M. H., W. J. Greenleaf , R. Landick, and S. M. Block. 2008. Applied force reveals mechanistic and energetic details of transcription termination. Cell, 132, 971-982.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: The principal output of research being conducted in this project are scientific publications. In addition, results of work were presented at a symposium in celebration of the Nobel Prize to Roger Kornberg. at Stanford, CA in April, 2007; at the FASEB Prok. Transcription Initiation Meeting at Saxtons River, VT in June, 2007; at a Biology in Motion conference organized by Cell Press in Evian Les Bains, france in October, 2007; and at a seminar presented at the Harvard Medical School in December 2007.
PARTICIPANTS: Principal Investigator: Robert Landick Researchers: Rachel Mooney, Jeff Grass, Murali Palangat, Jennifer Rowland Postoctoral Associates: Agata Czyz Graduate Research Assistants: Jason Peters, Kook Sun Ha, Jinwei Zhang
TARGET AUDIENCES: Biochemical, biophysical and molecular scientific research communities with the intent of increasing knowledge.
Impacts
Findings during the past year gave a completely new insight into the fundamental mechanism of RNA polymerase, the central enzyme of gene expression. These findings established that movements of a structural element called the trigger loop are responsible for allowing rapid RNA synthesis and open entirely new windows into our understanding of gene expression. In addition, results in studies of bacterial RNA polymerases have defined a new way to assay for novel inhibitors of RNA polymerase, which can serve as lead compounds for development of new antibiotics.
Publications
- Vassylyev, D. G.,, M. N. Vassylyeva, J. Zhang, M. Palangat, I. Artsimovitch, and R. Landick. 2007. Structural basis for substrate loading in bacterial RNA polymerase. Nature, 448, 163-168.
- Toulokhonov, I., J. Zhang, M. Palangat, and R. Landick. 2007. A central role of the RNA polymerase trigger loop in active-site rearrangement during transcriptional pausing. Mol. Cell, 27, 406-419.
- Davis, C. A., C. A. Bingman, R. Landick, M. T. Record, Jr.,and R. M. Saecker. 2007. Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase. Proc. Natl Acad. Sci. U.S.A., 104, 7833-7388.
- Kyzer, S. , K. Ha, R. Landick, and M. Palangat. 2007. Direct versus limited-step reconstitution reveals key features of an RNA hairpin-stabilized paused transcription complex . J. Biol. Chem., 282, 19020-19028.
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Progress 01/01/06 to 12/31/06
Outputs
Research in the Landick lab comprises a wide variety of topics related to RNA polymerase and the regulation of transcription. Regulation of Bacterial RNAP. Bacterial RNAP is regulated by intrinsic RNA/DNA signals that cause pausing or termination and by highly conserved elongation regulators such as Rho, NusA, and NusG. Key goals of current research are to determine where these regulators contact RNAP and how they alter its conformation. We investigate these issues using molecular genetics, protein-protein crosslinking and footprinting, rapid quench-flow kinetics, and protein engineering. A current high priority is to obtain a crystal structure of a paused transcription complex to learn how the active site is rearranged in the paused state. We also use single-molecule transcription assays with at Stanford and Brandeis, for which visualizing the effects of elongation regulators on RNAP movement is a high priority. Regulation of Human RNA Polymerase II Control of
transcript elongation plays central roles in human biology, for instance by regulating HIV-1 gene expression, controlling gene expression during differentiation (e. g., conversion of stem cells to specialized cells), and controlling mRNA splicing and processing, which are coupled to transcript elongation. However, the mechanisms by which elongation regulators control human RNAPII are largely unknown. We are investigating human RNAPII elongation control in several ways. First, we have developed rapid kinetic assays to dissect the elongation and pausing mechanism. Second, we are using site-directed mutagenesis to create specifically altered human RNAPII enzymes to investigate these mechanisms as well as how elongation regulators like DISF/NELF and TFIIS alter with RNAPII. Finally, we are developing single-molecule assays for human RNAPII so that we can exploit approaches similar to those we apply to bacterial RNAP. Study of Transcriptional Regulation in vivo We study transcription in
cells using two different approaches. In collaboration with Aseem Ansari (Biochemistry) and Tricia Kiley (Biomolecular Chemistry), we study the global distributions of RNAP and regulators in microbial cells growing in different conditions using so-called ChIP chip assays. These experiments promise to revolutionize our understanding of transcriptional regulation by showing how RNAP and regulators are distributed among genes as cells change their regulatory programs. We also are developing methods to produce recombinant RNAPs in E. coli from diverse bacteria, especially from difficult-to-study pathogens. We use the recombinant RNAPs to study their novel regulatory properties. This approach also is being developed as a high-throughput whole-cell assay for novel and lineage-specific inhibitors with potential applications as new antibiotics.
Impacts
Our research impacts basic understanding of the machinery of life by eluciating the structure of and function of RNA polymerase. It also has practical impacts in the development of new antibiotics and in improved ability to engineer gene expression.
Publications
- Ederth, J., R. A. Mooney, L. Issakson and R. Landick. 2006. Allosteric interplay between the downstream DNA jaw domain of bacterial RNA polymerase and allele-specific residues in the product RNA-binding pocket, J. Mol. Biol. 10, 1163-1179.
- Dalal R. V., M. H. Larson, K. C. Neuman, J. Gelles, R. Landick, and S. M. Block. 2006. Pulling on the nascent RNA during transcription does not alter kinetics of elongation or ubiquitous pausing. Mol. Cell 23, 231-239.
- Herbert, K. M. , A. La Porta, B. J. Wong, R. A. Mooney, K. C. Neuman, R. Landick, S. M. Block. 2006. Sequence-resolved detection of pausing by single RNA polymerase molecules. Cell 125, 1083-1094.
- Toulokhonov, I., and R. Landick. 2006. The role of the lid element in transcription by E. coli RNA polymerase, J. Mol. Biol. 361, 644-658.
- Landick, R. 2006. The regulatory roles and mechanism of transcriptional pausing. Biochem Soc. Transactions 34, 1062-1066.
- Landick, R. 2006. The Nobel prize for RNA polymerase - a long time in the making. Cell, 127, 1087-1090.
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