The DNA-dependent RNA polymerase play a central role in gene expression.
The aim of the proposed research is to produce a more precise genetic and biochemical description of the yeast RNA polymerase II. It is proposed that a combination of genetic and biochemical approaches are used to A) define the genuine components of RNA polymerase II, B) determine which subunits contribute to RNA catalysis and to interactions with promoters and transcription control factors, and C) identify transcription factors that interact directly with RNA polymerase II. To accomplish these goals, the specific aims of the experiments outlined in this proposal are: 1) to isolate and confirm the identity of the genes that specify each of the yeast RNA polymerase II subunits, by probing a recombinant DNA expression library with antibodies and by further characterization of the isolated genes. 2) to construct yeast mutants with lesions at defined RNA polymerase gene loci, resulting in conditional phenotypes, by using isolated subunit DNA sequences mutagenized in vitro to replace the wild-type gene. The conditional mutants constructed through this procedure will be used in an effort to: A) identify genuine RNA polymerase II subunits through biochemical analysis, B) identify subunits that interact with specific regulatory factors and examine the possibility that there exist multiple forms of RNA polymerase II, by analyzing the effects of the mutant alleles on the relative ability to express specific genes. 3) to isolated analyze and clone extragenic suppressors of the conditional RNA polymerase mutants in an effort to reveal RNA polymerase subunit interactions, previously undetected subunits and factors that interact with the enzyme. 4) to investigate the function of the carboxyl-terminal repeat of the large subunit of RNA polymerase II by constructing deletions in the repeat and assessing the effect on various steps in transcription. 5) to identify and isolate mutants in TATA box binding protein(s) by selecting for suppressors of a defective TATA box. Suppressing genes and their wildtype counterparts will be cloned to further study the putative protein(s). The health relatedness of this project derives from its contribution to the understanding of the basic molecular mechanisms which control gene expression.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034365-05
Application #
3285233
Study Section
Genetics Study Section (GEN)
Project Start
1984-12-01
Project End
1992-11-30
Budget Start
1988-12-01
Budget End
1989-11-30
Support Year
5
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Whitehead Institute for Biomedical Research
Department
Type
DUNS #
076580745
City
Cambridge
State
MA
Country
United States
Zip Code
02142
Ng, Huck Hui; Robert, Francois; Young, Richard A et al. (2003) Targeted recruitment of Set1 histone methylase by elongating Pol II provides a localized mark and memory of recent transcriptional activity. Mol Cell 11:709-19
Wyrick, J J; Aparicio, J G; Chen, T et al. (2001) Genome-wide distribution of ORC and MCM proteins in S. cerevisiae: high-resolution mapping of replication origins. Science 294:2357-60
Geisberg, J V; Holstege, F C; Young, R A et al. (2001) Yeast NC2 associates with the RNA polymerase II preinitiation complex and selectively affects transcription in vivo. Mol Cell Biol 21:2736-42
Robertson, L S; Causton, H C; Young, R A et al. (2000) The yeast A kinases differentially regulate iron uptake and respiratory function. Proc Natl Acad Sci U S A 97:5984-8
Kimmelman, J; Kaldis, P; Hengartner, C J et al. (1999) Activating phosphorylation of the Kin28p subunit of yeast TFIIH by Cak1p. Mol Cell Biol 19:4774-87
Hengartner, C J; Myer, V E; Liao, S M et al. (1998) Temporal regulation of RNA polymerase II by Srb10 and Kin28 cyclin-dependent kinases. Mol Cell 2:43-53
Woychik, N A; McKune, K; Lane, W S et al. (1993) Yeast RNA polymerase II subunit RPB11 is related to a subunit shared by RNA polymerase I and III. Gene Expr 3:77-82
McKune, K; Richards, K L; Edwards, A M et al. (1993) RPB7, one of two dissociable subunits of yeast RNA polymerase II, is essential for cell viability. Yeast 9:295-9
Choder, M; Young, R A (1993) A portion of RNA polymerase II molecules has a component essential for stress responses and stress survival. Mol Cell Biol 13:6984-91
Woychik, N A; Young, R A (1992) Genes encoding transcription factor IIIA and the RNA polymerase common subunit RPB6 are divergently transcribed in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 89:3999-4003

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