STRUCTURE &FUNCTION OF POL III TRANSCRIPTION FACTORS

Project: Research project

Project Details

Description

This application describes the use of a powerful genetic strategy
to target mutations in transcription factors and ancillary
components of the RNA polymerase 111 transcription apparatus of
yeast. This genetic approach circumvents problems encountered with
the biochemical purification of the transcription factors, TFIIIB
and TFIIIC, and provides a direct route to their genes. The
cloning of the genes for these factors and the determination of
their primary structures is the initial goal of this work.
Subsequent studies will identify sites of functional importance in
the cloned factors. Of particular interest are sites that are
engaged in protein-protein and protein-nucleic acid interactions
with other components of the transcription machinery. Finally,
studies are described in which the cloned transcription factor
genes are used to generate antibody probes and substrates to
investigate the posttramlational regulation of transcription factor
activity. The long term objectives of this work are (i) to obtain
a complete molecular description of the process of transcription
initiation and (ii) to gain an understanding of the mechanisms
responsible for regulating transcriptional activity and hence,
control of target gene expression. This system serves as a model
for higher eukaryotes and offers the advantages of combined
genetic, molecular biological and biochemical technologies to study
the primary event in gene expression, namely, the transcription of
RNA. Several genetic strategies, based on a common theme, are presented:
A unique tandem arrangement of tRNA nonsense suppressor genes is
described in which expression of a downstream (supS1) gene is
dependent upon transcription directed by the internal promoters of
an upstream (sup9-e) gene. Different promoter mutations in latter
prevent supS1 expression and thus provide a selection for
extragenic mutations that suppress the transcriptional defect.
Mutant strains have been isolated that suppress the effect of an
A-block (sup9-e A19) promoter mutation. Genetic characterization,
complementation analysis and a gene cloning protocol are described.
In vitro transcription experiments with whole-cell extracts and
fractionated transcription components from the mutant strains are
proposed as a means to identify the mutant factors and study their
mechanism of action. Studies on transcription factor structure-
function relationships and the regulation of transcription factor
activity will be conducted using these in vitro systems together
with in vivo and in vitro mutagenesis strategies.
StatusFinished
Effective start/end date12/31/893/31/00

Funding

  • National Institute of General Medical Sciences
  • National Institutes of Health
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences

ASJC

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

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