The mechanisms by which eukaryotes regulate gene expression are important for understanding many complex biological phenomena including human diseases. Prevention and treatment of such diseases have been and will continue to be improved by basic knowledge of gene regulation, especially because molecular mechanisms of transcriptional initiation are highly conserved in eukaryotic organisms ranging from human to yeast. This proposal will continue to investigate basic issues concerning molecular mechanisms of transcriptional initiation and elongation, polyadenylation, and mRNA stability in yeast, by combining molecular genetics, biochemical, functional genomic, and evolutionary approaches. First, we will address a variety of issues concerning transcriptional initiation and elongation including A) the nature and stability of the preinitiation and post-escape complexes and the role of Mediator in mediating transcriptional activation, B) the bi-directional nature of Pol II transcription and whether non-coding transcripts are functional or a mechanistic consequence of the transcription, C) whether Pol II pausing during elongation depends on DNA sequence of species-specific factors, D) the role of FACT and Spt6 in Pol II transcription and possible nucleosome eviction, and E) whether Pol II elongation factors travel in a large complex throughout the coding regions. Second, in the area of polyadenylation, we will A) address the mechanisms for why polyadenylation is restricted to the 3' UTR and identify factors that are responsible for the wild-type poly(A) pattern, B) identify the factor(s) that determines the different poly(A) site patterns in S. cerevisiae and D. hansenii, C) identify environmental conditions that cause differential polyadenylation and hence regulation of 3' isoforms, and D) identify factors important for regulated polyadenylation. Third, using our new approach to study mRNA decay, particularly of 3' isoforms, we will A) perform RNA structural analysis to provide direct evidence for our hypothesis that secondary structure involving the poly(A) tail and other regions play a key role in mRNA decay, B) address whether RNA secondary structure is determined by sequence or involves species-specific factors, C) identify protein factors mediating the large differences in mRNA stabilities D) perform directed genetic experiments to address how secondary structure affects mRNA stability, E) identify environmental conditions that cause differential mRNA decay and hence regulation of 3' isoforms, and F) identify factors important for regulated mRNA half-lives. Fourth, we will use a novel conceptual and experimental approach to distinguish biological function from biological noise that is based on a comparison of physiological responses, RNA and transcription factor binding profiles, and effects of mutations in yeast species of varying evolutionary distance. We will explicitly measure biological noise by making functional measurements of evolutionary irrelevant or random- sequence DNA in yeast. Overall, the proposal will answer fundamental questions about the interlinked processes of transcription, polyadenylation, and mRNA stability in a mechanistic and evolutionary framework.

Public Health Relevance

Regulation of gene expression is a critical aspect of many biological phenomena (e.g. cell growth, development of multicellular organisms, the response to environmental conditions, and evolution), and alteration of normal gene regulation can lead to human disease. This proposal investigates fundamental molecular mechanisms of gene regulation at several levels, and it also uses a novel functional evolutionary approach to understand broad issues related to biological function. The results will continue to shed new light on fundamental issues in gene regulation and will have a significant impact on our understanding of human biology and disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM030186-37
Application #
9543479
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Sledjeski, Darren D
Project Start
1982-03-01
Project End
2019-08-31
Budget Start
2018-09-01
Budget End
2019-08-31
Support Year
37
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Harvard Medical School
Department
Biochemistry
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
Moqtaderi, Zarmik; Geisberg, Joseph V; Struhl, Kevin (2018) Extensive Structural Differences of Closely Related 3' mRNA Isoforms: Links to Pab1 Binding and mRNA Stability. Mol Cell 72:849-861.e6
Jin, Yi; Eser, Umut; Struhl, Kevin et al. (2017) The Ground State and Evolution of Promoter Region Directionality. Cell 170:889-898.e10
Petrenko, Natalia; Jin, Yi; Wong, Koon Ho et al. (2017) Evidence that Mediator is essential for Pol II transcription, but is not a required component of the preinitiation complex in vivo. Elife 6:
Petrenko, Natalia; Jin, Yi; Wong, Koon Ho et al. (2016) Mediator Undergoes a Compositional Change during Transcriptional Activation. Mol Cell 64:443-454
Miotto, Benoit; Ji, Zhe; Struhl, Kevin (2016) Selectivity of ORC binding sites and the relation to replication timing, fragile sites, and deletions in cancers. Proc Natl Acad Sci U S A 113:E4810-9
Jin, Yi; Geisberg, Joseph V; Moqtaderi, Zarmik et al. (2015) Mapping 3' mRNA isoforms on a genomic scale. Curr Protoc Mol Biol 110:4.23.1-17
Wong, Koon Ho; Jin, Yi; Struhl, Kevin (2014) TFIIH phosphorylation of the Pol II CTD stimulates mediator dissociation from the preinitiation complex and promoter escape. Mol Cell 54:601-12
Moqtaderi, Zarmik; Geisberg, Joseph V; Struhl, Kevin (2014) Secondary structures involving the poly(A) tail and other 3' sequences are major determinants of mRNA isoform stability in yeast. Microb Cell 1:137-139
Geisberg, Joseph V; Moqtaderi, Zarmik; Fan, Xiaochun et al. (2014) Global analysis of mRNA isoform half-lives reveals stabilizing and destabilizing elements in yeast. Cell 156:812-24
Moqtaderi, Zarmik; Geisberg, Joseph V; Jin, Yi et al. (2013) Species-specific factors mediate extensive heterogeneity of mRNA 3' ends in yeasts. Proc Natl Acad Sci U S A 110:11073-8

Showing the most recent 10 out of 103 publications