Structural and Dynamic NMR Studies of RNA Polymerase

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Intellectual merit: The objective of this proposal is to use solution NMR spectroscopy to study RNAP in various transcriptional stages. The DNA-dependent RNA polymerase (RNAP) is the principal enzyme of gene expression and regulation in all cellular organisms. RNAP is a remarkable protein machinery capable of (i) specifically binding to promoter sites along the DNA, (ii) melting the double-stranded DNA (dsDNA) to form the transcription "bubble", and (iii) synthesizing the RNA chain complementary to the DNA template strand using nucleoside triphosphate (NTP) substrates. The essential core component of the bacterial RNAP (subunit composition alpha 2, beta, beta prime and omega) has been evolutionarily conserved from bacteria to humans. Sequence conservation points to structural and functional homologies, rendering the simpler bacterial RNAPs excellent model systems for understanding the basic principles at work for all cellular RNAPs. Recent progress in the structural, biochemical and biophysical characterization of RNAP has highlighted this enzyme as a complex, multifunctional protein machinery that functions by using an intricate balance of structural and dynamic changes. The goal of this project is to obtain integrated structural and dynamic information of the intricate mechanisms that underpin RNAP functionality by studying global and subtle structural changes as well as the amplitude and the time scale of functional motions. Towards this goal, the project will (1) develop strategies and methodologies to overcome the large size and complexity of RNAP, and (2) acquire structural and dynamic data on RNAP by applying an integrated NMR approach involving experiments tailored for (i) obtaining long-range structural information, (ii) detecting transiently populated conformational states, and (iii) determining both fast and slow time-scale motions and assessing their significance. Broader impact: Accomplishment of these objectives will have a tremendous impact on various fields. First, it will provide site-specific structural and dynamic information of RNAP during its action in solution, thereby offering unprecedented insight into the functional mechanisms of this important enzyme. Second, it will provide a model study about how NMR can be applied to obtain integrated structural and dynamic information on supramolecular biological systems. Third, it will establish NMR as a powerful tool for the dynamic characterization of large, intricate protein machineries by complementing static structures offered by X-ray crystallography. In addition to addressing fundamental biological questions, this project will be used to train students in structural biology, biophysics, and molecular biology, areas that are rapidly becoming integrated in 21st century science. Postdocs, graduate and undergraduate students will have the opportunity to be involved in a multi-disciplinary project that aims at the development of groundbreaking methodologies to enable characterization of supramolecular protein complexes by high resolution NMR spectroscopy. This will enable participants in the project to approach problems from a multidisciplinary and interactive perspective, thus experiencing first hand the utility of applying state-of-the-art methodologies to important biological problems. Two graduate students, supported by training grants and teaching assistantships, will do their theses on this project. They will be involved in the development of new labeling protocols and in the application of advanced NMR methodologies towards the dynamic and structural characterization of RNAP. The paradigm of combining structural, dynamic, thermodynamic and kinetic approaches to study complex protein systems will be included in a new course, currently designed by the PI, to exemplify the value of using an interdisciplinary and quantitative approach to answer questions of scientific and biomedical importance. The course is intended for a large, diverse audience consisting of graduate and advanced undergraduate students in the programs of Molecular Biosciences, Chemistry & Chemical Biology, Biomedical Engineering and BIOMAPS at Rutgers University.