Transcription is an essential process for life that constitutes the first step of gene expression in the cell and a crucial checkpoint for the control f critical biological events, including cell growth, differentiation, and oncogenesis. In eukaryotes, this highly regulated process is carried out by RNA polymerase II (Pol II). To transcribe the DNA, this enzyme must overcome the physical barriers imposed by the nucleosomal organization of the genetic material. Moreover, Pol II must transcribe torsionally constrained DNA segments that require the enzyme to translocate against increasing torques. Various mechanisms participate in the control and regulation of transcription, including structural changes of chromatin, various transcription factors, the polymerase ability to generate torque, and the activity of ATP-dependent chromatin remodeler complexes. In this application we propose to characterize these mechanisms of transcription control using a combination of single molecule manipulation and visualization approaches. Specifically, we propose: 1) Investigate the dynamics of transcription through nucleosomal arrays to elucidate the contribution of internucleosomal elements to the barrier. 2) Determine the mechanisms by which transcription factors IIS (TFIIS) and IIF (TFIIF) enhance nucleosomal transcription. 3) Characterize the relationship between the torsional strain in DNA and the ability of Pol II to transcribe through a nucleosome. 4) Characterize the nucleosome-remodeling mechanism, mechanochemical properties, and torque generation of the SWI/SNF complex.

Public Health Relevance

Oncogenesis and a large variety of developmental pathologies are associated with disruption of transcription, the central point of control for the regulation of gene expression. In this application we propose to characterize the dynamics of individual molecules of Pol II transcribing through arrays of nucleosomes, the effect of the enzyme's torque-generating capabilities on transcription throughput, the contribution of transcription factors to the ability of the enzyme to overcome the nucleosomal barrier, and the nucleosome remodeling mechanism of SWI/SNF. The insights that our experiments provide on transcription will lead to a better understanding of the basic molecular mechanisms of control of gene expression and they will support the first steps in the development of future therapies for transcription-based pathologies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM032543-32A1
Application #
8762266
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Lewis, Catherine D
Project Start
1983-07-01
Project End
2018-04-30
Budget Start
2014-08-01
Budget End
2015-04-30
Support Year
32
Fiscal Year
2014
Total Cost
$426,001
Indirect Cost
$136,001
Name
University of California Berkeley
Department
Miscellaneous
Type
Organized Research Units
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Pressé, Steve; Peterson, Jack; Lee, Julian et al. (2014) Single molecule conformational memory extraction: p5ab RNA hairpin. J Phys Chem B 118:6597-603
Dangkulwanich, Manchuta; Ishibashi, Toyotaka; Bintu, Lacramioara et al. (2014) Molecular mechanisms of transcription through single-molecule experiments. Chem Rev 114:3203-23
Liu, Tingting; Kaplan, Ariel; Alexander, Lisa et al. (2014) Direct measurement of the mechanical work during translocation by the ribosome. Elife 3:e03406
Kim, Hee-Kyung; Liu, Fei; Fei, Jingyi et al. (2014) A frameshifting stimulatory stem loop destabilizes the hybrid state and impedes ribosomal translocation. Proc Natl Acad Sci U S A 111:5538-43
Ishibashi, Toyotaka; Dangkulwanich, Manchuta; Coello, Yves et al. (2014) Transcription factors IIS and IIF enhance transcription efficiency by differentially modifying RNA polymerase pausing dynamics. Proc Natl Acad Sci U S A 111:3419-24
Bustamante, Carlos J; Kaiser, Christian M; Maillard, Rodrigo A et al. (2014) Mechanisms of cellular proteostasis: insights from single-molecule approaches. Annu Rev Biophys 43:119-40
Llorente-Garcia, Isabel; Lenn, Tchern; Erhardt, Heiko et al. (2014) Single-molecule in vivo imaging of bacterial respiratory complexes indicates delocalized oxidative phosphorylation. Biochim Biophys Acta 1837:811-24
Bintu, Lacramioara; Kopaczynska, Marta; Hodges, Courtney et al. (2011) The elongation rate of RNA polymerase determines the fate of transcribed nucleosomes. Nat Struct Mol Biol 18:1394-9
Kaiser, Christian M; Goldman, Daniel H; Chodera, John D et al. (2011) The ribosome modulates nascent protein folding. Science 334:1723-7
Cheng, Wei; Arunajadai, Srikesh G; Moffitt, Jeffrey R et al. (2011) Single-base pair unwinding and asynchronous RNA release by the hepatitis C virus NS3 helicase. Science 333:1746-9

Showing the most recent 10 out of 90 publications