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 #
1R01GM112686-01
Application #
8798014
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Preusch, Peter
Project Start
2015-01-15
Project End
2019-12-31
Budget Start
2015-01-15
Budget End
2015-12-31
Support Year
1
Fiscal Year
2015
Total Cost
$296,450
Indirect Cost
$103,950
Name
University of Pittsburgh
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Vergara, Sandra; Lukes, Dylan A; Martynowycz, Michael W et al. (2017) MicroED Structure of Au146(p-MBA)57 at Subatomic Resolution Reveals a Twinned FCC Cluster. J Phys Chem Lett 8:5523-5530
de la Cruz, M Jason; Hattne, Johan; Shi, Dan et al. (2017) Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED. Nat Methods 14:399-402
Kim, Sun Kyung; Barron, Lindsey; Hinck, Cynthia S et al. (2017) An engineered transforming growth factor ? (TGF-?) monomer that functions as a dominant negative to block TGF-? signaling. J Biol Chem 292:7173-7188
Shi, Dan; Nannenga, Brent L; de la Cruz, M Jason et al. (2016) The collection of MicroED data for macromolecular crystallography. Nat Protoc 11:895-904
Barnes, Christopher O; Kovaleva, Elena G; Fu, Xiaofeng et al. (2016) Assessment of microcrystal quality by transmission electron microscopy for efficient serial femtosecond crystallography. Arch Biochem Biophys 602:61-68
Stevenson, Hilary P; Lin, Guowu; Barnes, Christopher O et al. (2016) Transmission electron microscopy for the evaluation and optimization of crystal growth. Acta Crystallogr D Struct Biol 72:603-15
Baxter, Elizabeth L; Aguila, Laura; Alonso-Mori, Roberto et al. (2016) High-density grids for efficient data collection from multiple crystals. Acta Crystallogr D Struct Biol 72:2-11
Wu, Ying; Zhou, Xiaohong; Barnes, Christopher O et al. (2016) The DDB1-DCAF1-Vpr-UNG2 crystal structure reveals how HIV-1 Vpr steers human UNG2 toward destruction. Nat Struct Mol Biol 23:933-940
Barnes, Christopher O; Calero, Monica; Malik, Indranil et al. (2015) Crystal Structure of a Transcribing RNA Polymerase II Complex Reveals a Complete Transcription Bubble. Mol Cell 59:258-69