STRUCTURE AND FUNCTION OF POL III TRANSCRIPTION FACTORS

Project: Research project

Project Details

Description

The work described in this application exploits the genetic and
biochemical advantages offered by yeast to study a process of fundamental
importance in all eukaryotes, namely transcription by RNA polymerase III
(pol III). Pol III gene products participate in a myriad of essential
cellular processes including protein syntheses; pre-mRNA, pre-rRNA and
pre-tRNA maturation; protein transport and telomere synthesis. The
expression of pol III genes must be appropriate for the norma execution
of these processes and must be co-ordinated with cell growth. The long
term goal of this work is to obtain a detailed biochemical understanding
of how pol III genes are expressed and regulated at the transcriptional
level. To this end, a combination of molecular genetic and biochemical
approaches are being used to investigate the nature and function of
factors that play crucial but presently poorly defined roles in pol III
transcription.

a unique genetic strategy has yielded mutations in four genes (PCF1 to
PCF4), each of which increases transcription by pol III. The PCF1-1
mutation influences the rate of recruitment of transcription factor (TF)
IIIB to DNA. This effect will be examined by time-resolved footprinting.
Structure-function studies of PCF1 are also proposed in which new
dominant alleles and conditional mutations will be isolated. The latter
will be used to clone genes for interacting polypeptides by multi-copy
suppression.

Studies on PCF1-1 suggest that the binding of TFIIIB to DNA is
accompanied by a posttranslational event which activates a subunit of
this factor, TFIIIB90. Direct evidence that TFIIIB90 is
posttranslationally modified will be sought by in vivo labeling
experiments following the purification and cloning of this factor. Our
present transcriptional model suggests that PCF2-1 may encode the
catalytic activity responsible for TFIIIB90 activation. The nature and
function of PCF2 will be determined by gene sequencing and transcription
studies.

The PCF4-1 mutation identifies a polymerase specificity factor,
TFIIIB70, that is stoichiometrically limiting transcription. Experiments
are proposed to examine the effect of over-expressing this protein in
yeast and to study its interactions with other components of the
transcription machinery. Finally, a series of biochemical and molecular
experiments are outlined to test the hypothesis that PCF3, a negative
regulator of pol III transcription, mediates growth control over this
process.
StatusFinished
Effective start/end date7/1/946/30/95

Funding

  • National Institute of General Medical Sciences

ASJC

  • Genetics
  • Molecular Medicine
  • Molecular Biology
  • Cell Biology
  • Microbiology

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