RNAP ALPHA--DOMAIN ORGANIZATION, STRUCTURE &DNA BINDING

Project Details

Description

Escherichia coli RNA polymerase has subunit composition
(alpha)2BetaBetasigma. RNA polymerase alpha subunit carries out three
important functions (i) alpha serves as the initiator for RNA polymerase
assembly, (ii) alpha participates in promoter recognition by direct
sequence-specific protein-DNA interaction, and (iii) alpha is the target
for a large set of transcription activator proteins, including catabolite
gene activator protein (CAP). Our research project on RNA polymerase alpha subunit is directed at four
long-range objectives: (i) determination of the three-dimensional
structure of RNA polymerase, (ii) understanding promoter recognition,
(iii) understanding transcription initiation, and (iv) understanding
transcription activation. Our research project has three specific aims: Specific Aim 1: Analysis of domain organization of alpha. Experiments
are proposed to dissect a into protease-resistant protein fragments
("domains), to determine the N-terminal and C-terminal boundaries of
alpha domains, and to analyze the oligomerization properties, assembly
properties, and DNA binding properties of alpha domains. Specific Aim 2: Analysis of structure of alpha and alpha domains.
Experiments are proposed to prepare gram quantities of alpha and alpha
domains, to determine secondary structure compositions by CD
spectroscopy, and to determine tertiary structures by multi-dimensional
NMR spectroscopy and x-ray crystallography. Specific Aim 3: Analysis of DNA binding by alpha. Experiments are
proposed to develop preoperative and quantitative assays for alpha-DNA
interaction; to determine the "optimal" DNA-site sequence and minimum
DNA-site length for alpha-DNA interaction; to quantify the DNA binding
stoichiometry, DNA binding affinity, and DNA binding specificity of 4
alpha-DNA interaction; to identify DNA binding residues by site-specific
5-bromouracil-mediated photocrosslinking, site-specific aldehydic-abasic-
site-mediated crosslinking, and alanine-scanning mutagenesis; and to
analyze the role of alphaDNA interaction in transcription initiation. The results to be obtained will be directly relevant to understanding
regulation of prokaryotic gene expression. Since eukaryotic RNA
polymerase subunits share sequence and mechanistic homologies with E.
coli RNA polymerase subunits, including a, the results to be obtained
also may be relevant to understanding regulation of eukaryotic gene
expression.
StatusFinished
Effective start/end date8/1/941/31/99

Funding

  • National Institutes of Health: $269,033.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $273,818.00

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

Fingerprint Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.