Project Details
Description
Careful orchestration of mRNA synthesis is critical for programmed cell
death, normal cell growth and development, and maintenance of health and
well-being in humans. The broad, long term objectives of this project are
to understand the mechanisms that underlie the intricate regulation of
eukaryotic gene expression. Understanding how gene expression occurs in a
timely and coordinate manner requires detailed knowledge of the components
of the transcriptional machinery. The heart of this machinery comprises the
RNA polymerase II holoenzyme, a multiprotein complex that contains RNA
polymerase II. This proposal focuses on the study of five of the holoenzyme
components, the RNA polymerase II subunits designated RPB4, RPB5, RPB6,
RPB7 and RPB9, to help identify their roles in transcription. Specific Aim
l involves making conditional mutants in each of these subunits and testing
them for transcriptional defects using several in vivo and in vitro
assays.- In Specific Aim II, mutants affecting initiation, elongation or
termination will be subjected to suppressor analysis in order to uncover
novel interacting proteins and identify known transcription factors that
directly contact RNA polymerase. To complement this genetic approach, two-
hybrid screens with the wild-type subunits will be used to identify
additional interacting proteins that may not be revealed by genetic
suppression experiments. In Specific Aim III the biological significance
of RPB6 phosphorylation will be investigated. Potential phosphorylation
sites at ten conserved residues will be altered using site-directed
mutagenesis. The mutants will be subjected to in vitro transcription
experiments and other in vivo studies. Phosphorylation site-specific
antibodies will also be enlisted to help pinpoint the location of the
target site. Finally, kinases that are involved in RPB1-carboxyl terminal
domain phosphorylation and other candidate kinases will be tested for
potential roles in RPB6 phosphorylation and function.
Status | Finished |
---|---|
Effective start/end date | 5/1/97 → 4/30/03 |
Funding
- National Institute of General Medical Sciences: $252,674.00
- National Institute of General Medical Sciences: $250,931.00
ASJC
- Genetics
- Molecular Biology
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