BIOCHEMISTRY OF EUKARYOTIC TRANSCRIPTION REGULATION

Project: Research project

Project Details

Description

A large combinatorial assortment of DNA sequence-specific transcriptional
activators control the expression of the vast repertoire of eukaryotic
cellular and viral genes, which in turn dictate the biochemistry of the
cell. How the multitude of activators funnel their signal into a common
target, RNA polymerase II which transcribes these genes, is currently
unknown. Solving this unknown would provide a major step toward
understanding the biochemistry of cellular development differentiation,
oncogenesis, and viral infectivity. This project proposes to chip away at the unknown biochemistry of
transcriptional regulation by investigating the molecular mechanism through
which one transcriptional activator, human Sp1, communicates with the human
transcription machinery. In vitro assays which reconstitute Sp1-regulated
transcription with purified components are already in place. The factors
which Sp1 targets in the initiation complex have been localized to a
purified multi-subunit TFIID complex. TFIID is a key component of a larger
RNA polymerase II transcription initiation machine assembled at the
promoter. The next step is to biochemically characterize purified intact
TFIID complexes and their individual subunits (TAFs). Differences in
subunit composition are proposed to generate multiple TFIID complexes, each
with different gene regulatory functions. TFIID complexes have been and
will be purified by immuno-affinity chromatography using antibodies directe
against its TATA box binding subunit (TBP). First, the number of
chromatographically distinct TFIID complexes and their subunit composition
will be assessed by silver stained SDS polyacrylamide gels. Second,
denaturants such as urea will be used to fractionate purified TAF subunits
from each other, which will then be biochemically characterized. One or
more TAFs comprise the SP1 coactivator which is necessary for Sp1-activated
but not basal transcription. The purified coactivator will be reconstitute
and its role in assembling an Sp1-regulated initiation complex addressed by
DNase I protection, electrophoretic mobility shift, and kinetic assays.
These assays will allow the following questions to be addressed: What rate
limiting step in initiation does Sp1 target? Are on/off rates of limiting
initiation factor affected? Does Sp1 target and mollify an inhibitory
activity in the TFIID complex? An unusual feature of Sp1 is its ability to activate transcription at
promoters which lack a TATA box. At such promoters an additional component
of the TFIID complex, termed a tethering factor, is essential for Sp1-
activated transcription; at TATA-containing promoters, Sp1-activated
transcription does not require the tethering factor. The tethering factor
does not appear to recognize the promoter directly through protein-DNA
interactions, but is proposed to achieve promoter specificity through
protein-protein interactions with Spl. AS with the coactivator the
tethering factor will be isolated, cloned, and its role in assembling an
initiation complex at a TATA-less promoter investigated. It is anticipated
that information gained from this study will provide an essential missing
piece to the gene regulation puzzle.
StatusFinished
Effective start/end date8/1/923/31/99

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health

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