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> | Abstract: DESCRIPTION (provided by applicant) Transcription by RNA polymerase (pol) III is of fundamental importance in all eukaryotes since its products, which include 5S RNA, tRNA, U6 snRNA, RNase P RNA and 7SL RNA, are essential for protein synthesis, RNA processing, protein transport and other cellular processes. The transcription of pol III genes is tightly coupled with cell growth and is co-regulated with transcription of the large ribosomal RNAs by pol I. These transcriptional processes account for about 80% of nuclear transcription in growing cells and their coordinate regulation is thought to be important for metabolic economy and biological fitness. The majority of the proteins that comprise the pol Ill transcription machinery are conserved from yeast to humans. Thus, in almost all cases, the knowledge obtained from studies on pol III transcription in yeast is readily translated into human cells. In higher eukaryotes, pol III transcription is activated by mutations in tumor suppressor genes (p53 and RB) and by cell transformation with viral and cellular oncogenes. It is thought, therefore, that the elevated growth rate of transformed cells is dependent, in part, on their high levels of pol III transcription. The identities of the pol III transcription components that are subject to regulation under a variety of conditions and the mechanisms of their regulation are not well-defined in any system. Accordingly, the long-term objectives of this research are to identify the regulatory targets in the pol III system and determine the ways in which their function is controlled. To this end, the experiments in this application will provide a detailed biochemical understanding of a limiting step in the concerted assembly of the pol III initiation factor TFIIIB in Saccharomyces cerevisiae, namely the interaction between the tetratricopeptide repeat (TPR)- containing subunit of TFIIIC (TFIIIC131) and TFIIB-related subunit of TFIIIB (Brf1). This study will also serve as a valuable paradigm for understanding the binding specificity and function of the ubiquitous TPR motif in the assembly of multi-subunit complexes. In addition, the experiments will identify the pol III factors that respond to upstream signaling pathways and mediate transcriptional repression in yeast. These factors will be examined using biochemical and molecular genetic methods to elucidate the molecular mechanisms of their regulation. The hypotheses developed in course of these studies will be explicitly tested by a combination of in vivo and in vitro assays.
Public Health Relevance:
This Public Health Relevance is not available.
Thesaurus Terms:
DNA directed RNA polymerase, cell growth regulation, genetic regulation, genetic transcription, protein structure function, transcription factor
binding site, cell cycle, fungal genetics, fungal protein, gene induction /repression, molecular genetics, mutant, nucleic acid repetitive sequence, p53 gene /protein, posttranslational modification, protein protein interaction, protein sequence, suppressor mutation
SDS polyacrylamide gel electrophoresis, Saccharomyces cerevisiae, microorganism culture, polymerase chain reaction, western blotting
Institution: YESHIVA UNIVERSITY
500 W 185TH ST
NEW YORK, NY 10033
Fiscal Year: 2008
Department: BIOCHEMISTRY
Project Start: 01-JUL-1989
Project End: 30-NOV-2009
ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
IRG: BIO
Grant Number: 1R01GM085177-01A1
Project Title: Transcriptional Repression by Maf1 in Yeast
PI Information: Name Email Title
WILLIS, IAN M. willis@aecom.yu.edu PROFESSOR
Abstract: DESCRIPTION (provided by applicant): The synthesis of rRNAs and tRNAs is intimately linked with cell division through the function of tumor suppressors (e.g. p53 and Rb) and oncogenes (e.g. c-myc). The transforming forms of these proteins directly target and up-regulate transcription by RNA polymerases (pols) I and III along with the expression of important cell cycle regulators. The Maf1 protein is a unique regulator of transcription whose properties in human cells are similar to p53 and Rb. Human Maf1 is a candidate tumor suppressor that negatively regulates transcription by all three nuclear RNA polymerases. How Maf1 achieves its effects on transcription is not understood and is complicated by the fact that the protein does not contain any motifs of known function. In S. cerevisiae, Maf1 is essential for repressing transcription by RNA polymerase III and functions to integrate the responses from multiple nutritional and stress signaling pathways that coordinately regulate ribosome and tRNA synthesis. These pathways, like Maf1 itself, are not well defined. The universal requirement for Maf1 in pol III transcriptional repression in yeast provides an extraordinarily valuable model for understanding the pathways and mechanisms regulating ribosome and tRNA synthesis and the biochemical function of this novel signal integrator. Accordingly, the long-term goal of this research is to understand Maf1 structure/function relationships, its interactions with regulatory targets and its interactions with upstream signaling molecules that operate via posttranslational mechanisms to affect Maf1-dependent transcriptional repression. These aims will be achieved through biochemical studies in well-defined in vitro systems and through the application of an innovative method for phosphopeptide identification. In addition, powerful genome-wide and systematic genetic and biochemical approaches will be used to identify protein kinases and other molecules involved in signaling repression by Maf1. Finally, our understanding of the function and regulation of Maf1 will be advanced by the determination its three-dimensional structure. PUBLIC HEALTH RELEVANCE: Maf1 is a potential tumor suppressor that negatively regulates transcription by all three nuclear RNA polymerase in mammalian cells. In yeast, Maf1 is an essential mediator of transcriptional repression by RNA polymerase III and integrates the responses from multiple nutritional and stress signaling pathways that coordinately regulate ribosome and tRNA synthesis. Our genetic, biochemical and structural studies on on Maf1 will enhance understanding of fundamental cellular processes that are likely to impact cancer biology.
Public Health Relevance: Maf1 is a potential tumor suppressor that negatively regulates transcription by all three nuclear RNA polymerase in mammalian cells. In yeast, Maf1 is an essential mediator of transcriptional repression by RNA polymerase III and integrates the responses from multiple nutritional and stress signaling pathways that coordinately regulate ribosome and tRNA synthesis. Our genetic, biochemical and structural studies on on Maf1 will enhance understanding of fundamental cellular processes that are likely to impact cancer biology.
Thesaurus Terms:
There are no thesaurus terms on file for this project.
Institution: YESHIVA UNIVERSITY
500 W 185TH ST
NEW YORK, NY 10033
Fiscal Year: 2009
Department: BIOCHEMISTRY
Project Start: 01-MAR-2009
Project End: 28-FEB-2013
ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
IRG: MGC |
