Regulation of elongation by RNA Polymerase II (Pol II) is less well understood than initiation and has wide- ranging importance for gene expression and chromatin structure. Pol II elongation in eukaryotes has several main functions. Of primary importance, Pol II is responsible for expression of protein-coding gene and certain essential snRNA genes. Pol II transcription, including the elongation phase, has wide-ranging impacts on chromatin structure and composition due to cotranscriptional chromatin remodeling and modification. A low level of transcription is pervasive throughout eukaryotic genomes, revealing new challenges to the regulation and control of proper gene expression. A large number of factors have been linked to Pol II transcription during the elongation phase, but their roles in the maintenance and control of Pol II elongation are relatively unknown. The proposed research will identify the mechanisms and regulation of Pol II elongation in vivo using the structurally, genetically and molecularly amenable Saccharomyces cerevisiae. The research builds on the previous identification of a critical role for the highly conserved, conformationally flexible trigger loop (TL) of the Pol II large subunit, Rpb1, in substrate selection, control of elongation rate and as the direct target for the Pol II-elongation specific inhibitor, 1-amanitin. We have generated a series of Pol II mutants with alterations in just a few trigger loop residues that have differences in elongation rate over two orders of magnitude in vitro. In the current work, these mutants with a range of Pol II rates will be used to dissect mechanisms of Pol II elongation in vivo using a multipronged approach. First, it will be determined how mutations that alter Pol II elongation rate in vitro affect Pol II processivity and elongation rate in vivo using chromatin IP, detection of nascent RNA and transcription run on. Second, the consequences of alteration of Pol II activity by substrate limitation in vivo will be determined using a novel genetic system. This system will allow testing of a long standing but unproven phenotype that has been used for the putative identification of elongation factors. Third, a specific model will be tested for Pol II elongation contribution to initiation through the efficiency of promoter escape, wherein transcription begins but may be unsuccessful in reaching a mature elongation phase. This model has broad implications for how elongation factors might control transcription, and how promiscuous initiation may be normally limited. Finally we will determine at nucleotide resolution effects on Pol II pausing, distribution, transcription start site selection and transcriptome content in response to alteration of Pol II TL function. These experiments will reveal the nature of elongation defects in vivo, and how alteration of elongation contributes to gene expression. This work will establish new paradigms for the dissection of Pol II elongation factor function.

Public Health Relevance

This project aims to uncover basic mechanisms for gene expression in a model eukaryotic organism, Baker's yeast. The proteins responsible for control of gene expression in Baker's yeast are highly related to proteins with the same functions in human cells. Precise control of gene expression is required for proper human development and prevention of disease. Our studies will directly impact the understanding of gene expression in humans, and thus are relevant to human health.!

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM097260-02
Application #
8320226
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Sledjeski, Darren D
Project Start
2011-08-15
Project End
2016-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
2
Fiscal Year
2012
Total Cost
$270,722
Indirect Cost
$81,972
Name
Texas A&M University
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
078592789
City
College Station
State
TX
Country
United States
Zip Code
77845
Schweikhard, Volker; Meng, Cong; Murakami, Kenji et al. (2014) Transcription factors TFIIF and TFIIS promote transcript elongation by RNA polymerase II by synergistic and independent mechanisms. Proc Natl Acad Sci U S A 111:6642-7
Pai, Dave A; Kaplan, Craig D; Kweon, Hye Kyong et al. (2014) RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template. RNA 20:644-55
Cabart, Pavel; Jin, Huiyan; Li, Liangtao et al. (2014) Activation and reactivation of the RNA polymerase II trigger loop for intrinsic RNA cleavage and catalysis. Transcription 5:
Kaplan, Craig D (2013) Basic mechanisms of RNA polymerase II activity and alteration of gene expression in Saccharomyces cerevisiae. Biochim Biophys Acta 1829:39-54
Braberg, Hannes; Jin, Huiyan; Moehle, Erica A et al. (2013) From structure to systems: high-resolution, quantitative genetic analysis of RNA polymerase II. Cell 154:775-88
Viktorovskaya, Olga V; Engel, Krysta L; French, Sarah L et al. (2013) Divergent contributions of conserved active site residues to transcription by eukaryotic RNA polymerases I and II. Cell Rep 4:974-84
Kaplan, Craig D; Jin, Huiyan; Zhang, Ivan Liang et al. (2012) Dissection of Pol II trigger loop function and Pol II activity-dependent control of start site selection in vivo. PLoS Genet 8:e1002627