DNA-directed RNA Polymerase II (Pol II) is one of the most important proteins in the cell. Pol II is responsible for transcribing the vast majority of genes to generate messenger RNA that will be translated in the ribosomes to produce all cellular proteins. The initiation stage of transcription requires timely interactions between Pol I and the general transcription factors or GTFs including, TFIIB, TBP, TFIIF, TFIIH and TFIIE. The process of initiation is highly dynamic; biochemical experiments hint at possible discrete stages where the GTFs recognize, melt and load a nucleic acid scaffold. Initial promoter melting is triggered by TFIIH helicases that generate a 7-9 bases transcription bubble (TB); the bubble is further unwound to approximately 18-25 bases and descends to Pol II's active site where a short DNA-RNA hybrid (transcript) is synthesized; transcripts of 10 or more nucleotides result in promoter escape and stabilization of a mature TB that ultimately leads to dislodging of the GTFs leaving a poised Pol II for entry into productive elongation. This process is universal, for all eukaryotic species, and is at the core of gene regulation; understanding its molecular details will provide essential clues that could potentially lead to pharmacological manipulation of gene expression. The intention of this proposal is to use biochemical and X-ray crystallography techniques to understand the molecular details of promoter binding to Pol II and the role that TFIIB and TFIIF play in TB loading and stabilization.

Public Health Relevance

DNA-directed RNA Polymerase II (Pol II) is one of the most important proteins in the cell. Pol II is responsible for transcribing the vast majority of genes t generate messenger RNA that will be translated in the ribosomes to produce all cellular proteins. The initiation stage of transcription requires timely interactions between Pol II and the general transcription factors (TFIIB, TBP, TFIIF, TFIIH and TFIIE). The process of initiation is highly dynamic; biochemical experiments hint at possible discrete stages where the GTFs recognize, melt and load a nucleic acid scaffold (NAS). This mechanism is universal, for all eukaryotic species, and is at the core of gene regulation; understanding its molecular details will provide essential clues that could potentially lead to pharmacological manipulation of gene expression. The intention of this proposal is to study the early stages of transcription initiation using X-ray crystallographic techniques.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM112686-05
Application #
9616274
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Sledjeski, Darren D
Project Start
2015-01-15
Project End
2019-12-31
Budget Start
2019-01-01
Budget End
2019-12-31
Support Year
5
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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