The goal of the proposed research is a complete description of mRNA synthesis at the near atomic level. RNA chain elongation, once regarded as a matter of monotonous ribonucleotide addition, is now known to comprise a complex interplay of RNA polymerase states and interacting protein factors. We have previously determined the X-ray structures of pre- and post-translocated states, leading to the trigger loop hypothesis for specificity and catalysis in the post-translocated state. We now propose a combination of genetic and biochemical studies, as well as extension to higher resolution, to evaluate this hypothesis and fully illuminate the catalytic mechanism. We will also determine the remaining RNA polymerase structures, those of paused and arrested backtracked states. Structures of cocrystals with auxiliary protein factors will reveal the basis for transitions between states, for proofreading the transcript in the paused state, for recovery from the arrested state, and for transcriptional regulation.
Aims for the next project period are as follows: 1. Determine structures of transcribing complexes containing RNA polymerase mutations and nucleotide analogs. Polymerase mutants will be derived from a comprehensive genetic screen, which identifies amino acid residues important for transcription elongation in vivo. Nucleotide analogs will vary in size, shape, and hydrogen-bonding capability. Resolution will be extended with better crystals, data collection on microfocus beamlines, and improved data processing. The results will test the trigger loop hypothesis and give detailed insight into the transcription mechanism. 2. Determine structures of backtracked complexes. Short lengths of backtracked RNA will give rise to paused complexes, and longer backtracked RNAs will recreate the arrested state. The results will test hypotheses for interaction of the longer RNAs with the polymerase and for the control of backtracking. 3. Determine structures of cocrystals with TFIIS, Spt4/5, and Ubc5/Rsp5. TFIIS provokes the cleavage of backtracked RNAs, resulting in proofreading of paused complexes and recovery from the arrested state. Spt4/5 plays a key role in the regulation of transcription through promoter-proximal pausing. Ubc5/Rsp5 couple arrest to the ubiquitylation and destruction of RNA polymerase II.

Public Health Relevance

The significance of the proposed research may be summarized as follows: it will provide the structural information needed to fully understand the fundamental mechanism of transcription;it will establish a structural basis for studies of transcriptional regulation;and it will facilitate the design of new therapies for diseases of aberrant gene regulation in the future.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM049985-18
Application #
8027756
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Flicker, Paula F
Project Start
1993-08-01
Project End
2012-12-31
Budget Start
2011-01-01
Budget End
2011-12-31
Support Year
18
Fiscal Year
2011
Total Cost
$557,479
Indirect Cost
Name
Stanford University
Department
Biology
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Robinson, Philip J; Trnka, Michael J; Bushnell, David A et al. (2016) Structure of a Complete Mediator-RNA Polymerase II Pre-Initiation Complex. Cell 166:1411-1422.e16
Lu, Jonathan; Trnka, Michael J; Roh, Soung-Hun et al. (2015) Improved Peak Detection and Deconvolution of Native Electrospray Mass Spectra from Large Protein Complexes. J Am Soc Mass Spectrom 26:2141-51
Murakami, Kenji; Mattei, Pierre-Jean; Davis, Ralph E et al. (2015) Uncoupling Promoter Opening from Start-Site Scanning. Mol Cell 59:133-8
Guan, Shenheng; Trnka, Michael J; Bushnell, David A et al. (2015) Deconvolution method for specific and nonspecific binding of ligand to multiprotein complex by native mass spectrometry. Anal Chem 87:8541-6
Murakami, Kenji; Tsai, Kuang-Lei; Kalisman, Nir et al. (2015) Structure of an RNA polymerase II preinitiation complex. Proc Natl Acad Sci U S A 112:13543-8
Fazal, Furqan M; Meng, Cong A; Murakami, Kenji et al. (2015) Real-time observation of the initiation of RNA polymerase II transcription. Nature 525:274-7
Azubel, Maia; Koivisto, Jaakko; Malola, Sami et al. (2014) Nanoparticle imaging. Electron microscopy of gold nanoparticles at atomic resolution. Science 345:909-12
Liu, Xin; Bushnell, David A; Kornberg, Roger D (2013) RNA polymerase II transcription: structure and mechanism. Biochim Biophys Acta 1829:2-8
Robinson, Philip J J; Bushnell, David A; Trnka, Michael J et al. (2012) Structure of the mediator head module bound to the carboxy-terminal domain of RNA polymerase II. Proc Natl Acad Sci U S A 109:17931-5
Liu, Xin; Bushnell, David A; Silva, Daniel-Adriano et al. (2011) Initiation complex structure and promoter proofreading. Science 333:633-7

Showing the most recent 10 out of 19 publications