. The long-term goal of this project is to understand how transcription by RNA polymerase II (RNApII) is coupled to RNA processing and termination. Earlier funding periods of this project produced a model in which the C-terminal domain (CTD) of the RNApII subunit Rpb1 displays characteristic phosphorylation patterns at different stages of the transcription cycle to promote binding of the appropriate factors for co-transcriptional RNA processing. These studies are done in the model system Saccharomyces cerevisiae, but this process is highly conserved over evolution. The fundamental knowledge generated by this project provides significant insight into how the CTD phosphorylation cycle affects medically important processes such as the stimulation of HIV transcription by the viral Tat protein and the "pausing" of RNApII at developmentally regulated genes in embryonic stem cells. Continuation of this project is necessary to better understand both the enzymes that mediate the changes in CTD phosphorylation (kinases, phosphatases, etc.) as well as the proteins that recognize these patterns. In the next funding period, four specific aims are proposed. The first is a continuation of studies to understand the mechanisms of the two known termination pathways and how RNApII chooses between them: an early termination pathway used for small noncoding transcripts and many "cryptic" transcripts or the later polyadenylation-coupled termination pathway used for mRNAs. A second related aim is to study the biogenesis of short unstable transcripts produced by divergent transcription of many RNApII promoters, with a focus on what they can tell us about initiation, early elongation, and termination.
The third aim will use a new, high- resolution technique for mapping 3'ends of nascent transcripts to further probe the role of CTD phosphorylation in RNA elongation, pausing, and termination. The fourth specific aim builds upon the surprising discovery that the Rpb1 CTD can be transferred onto a different RNApII subunit and still function. This finding forms the basis for a set of experiments asking whether the different CTD modifications and functions need to occur in "cis" on the same CTD or can be split between two CTDs, and whether these phosphorylations can be bypassed by tethering their targets directly to RNApII.

Public Health Relevance

Improper gene expression causes many diseases, including developmental defects and cancer. The goal of this project is to understand the fundamental processes by which genes are expressed and regulated. This understanding will be essential for designing treatments and drugs to restore normal gene expression in diseased cells or to alter gene expression patterns to create pluripotent stem cells and specific differentiated cell types.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM056663-15
Application #
8479366
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Sledjeski, Darren D
Project Start
1999-07-01
Project End
2015-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
15
Fiscal Year
2013
Total Cost
$413,257
Indirect Cost
$167,069
Name
Harvard University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
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Lunde, Bradley M; Reichow, Steve L; Kim, Minkyu et al. (2010) Cooperative interaction of transcription termination factors with the RNA polymerase II C-terminal domain. Nat Struct Mol Biol 17:1195-201

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