ASSEMBLY OF POLYMERASE SPECIFIC TRANSCRIPTION COMPLEXES

Project: Research project

Project Details

Description

Accurate transcription by each of the three eukaryotic nuclear RNA
polymerases (pols) requires the assembly and recognition of unique
transcription complexes on the promoters of appropriate genes. These
complexes have at least one common component, the TATA box binding protein
(TBP). In addition, complexes recognized by pols Il and III contain
homologous proteins (TFIIB and TFIIIB70) that are known or believed,
respectively, to interact directly with TBP. The properties of these
molecules raise an important question: What mechanisms exist to ensure
that a transcription complex with the correct polymerase-specificity will
assemble on a particular gene? The long term goal of this proposal is to
describe in quantitative terms, the biochemical basis for the assembly of
RNA polymerase-specific transcription complexes.

Initially, we propose to carry out quantitative analyses of the
thermodynamics and kinetics of TBP-TFIIB and TBP-TFIIIB70 associations
with model pol II and pol III promoters. Specially adapted enzymatic and
chemical protection methods will be used for these studies. We will also
analyze the assembly of TBP-TFIIIB70 and TBP-TFIIB complexes in solution
by analytical ultracentrifugation. These experiments will be facilitated
by using spectroscopically labeled TBP. Together, these studies will
define quantitatively the chemical interactions that characterize the
assembly of RNA polymerase-specific complexes.

In the second phase of the work, spectroscopically-labeled transcription
factors will be used to follow conformational changes of the TBP-TFIIB and
TBP-TFIIIB70 complexes in solution and after binding to specific DNA
templates. In the latter case, conformation changes to the DNA will also
be followed using appropriate chemical and enzymatic probes. We will then
correlate the conformational changes in the proteins and the DNA with
specific kinetic or binding events resolved in the initial phase of the
proposal. These data will provide knowledge of the mechanistic
differences underlying the unique specificity of the complexes.
StatusFinished
Effective start/end date8/1/947/31/95

Funding

  • National Institute of General Medical Sciences

ASJC

  • Molecular Biology
  • Structural Biology
  • Biophysics
  • Biochemistry
  • Inorganic Chemistry

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