CHEMICAL PROBES OF RNA POLYMERASE AND DNA

  • Meares, Claude F, (PI)

Project: Research project

Description

Understanding gene expression in bacteria has produced health benefits
ranging from biotechnology to cancer therapy. The molecular mechanism of
gene transcription can be studied with high resolution by techniques that
share a common basis in chemistry. Previously, we have designed
experiments that identify the macromolecules contacted by the leading end
of nascent RNA at almost every step of its path through the transcription
complex which catalyzes its synthesis. The next task is to understand the
number and nature of the binding sites that form the RNA path. We propose
to apply new methods to identify the individual residues in the proteins
and DNA contacted by nascent RNA as it passes through the transcription
complex, providing a wealth of information about the process. 1. Using an artificial protease---a small metal chelate that can be
activated when desired, which we conceived and developed during the
current period---we shall map those amino-acid residues in the enzyme
subunits that come into contact with the leading end of the nascent RNA
molecule. This will be done by preparing complexes containing defined
lengths of RNA without protease at the leading end, and then activating
the protease. Our protease works by proximity, not by residue type; it
selectively cleaves peptide bonds directly adjacent to it. Isolation of
the new fragments and sequencing around the cleavage sites will provide a
high resolution map of those amino acid residues forming the path of RNA
across the enzyme's surface, along with their positions in the primary
structures of the enzyme subunits involved. Recent advances in the
technology of preparing stable transcription complexes containing defined
lengths of RNA have significantly altered our experimental protocols. We
shall investigate and compare transcription complexes made (i) using
dinucleotide initiators and selected chain terminators and (2) using
artificial transcription bubbles made from machine-synthesized nucleic
acids. 2. We shall determine the RNA lengths and DNA sites at which the leading
end of the transcript contacts the DNA template in transcription
complexes. Several promoters will be compared, since the DNA sequence is
likely to be an important factor. This will be carried out (1) using an
aromatic azide at the leading end of a dinucleotide primer, producing a
collection of terminated transcription complexes, photolyzing it and
isolating and characterizing the photo-crosslinked RNA/DNA conjugate; and
(2) using limited elongation to prepare a set of artificial transcription
bubbles each with a probe attached to the leading end of RNA.
StatusFinished
Effective start/end date12/1/786/30/07

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $300,955.00
  • National Institutes of Health: $161,503.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $236,998.00
  • National Institutes of Health
  • National Institutes of Health: $236,634.00
  • National Institutes of Health
  • National Institutes of Health: $221,920.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $165,058.00
  • National Institutes of Health

Fingerprint

DNA-Directed RNA Polymerases
RNA
DNA
DNA Probes
Nucleic Acids
Transcription
Macromolecules
Proteins
Thiouracil
Prokaryotic Cells
Escherichia coli
Sigma Factor
Enzymes
RNA Polymerase II
Insurance Benefits
Peptides
Ribosomes
Genes
Peptide Hydrolases
Gene Expression

ASJC

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