The long-term objective of this project is to understand how eukaryotic cells regulate gene expression at the level of transcription elongation. Eukaryotes regulate the expression of many genes including c-myc, c-myb, and c-fos by controlling the ability of RNA polymerase II to synthesize complete primary transcripts. Chromosomal rearrangements in Burkitt's lymphoma are associated with the derangement of c-myc's regulated block to elongation. The biochemical basis for regulating transcription elongation in animal cells is unknown and the RNA polymerase II elongation complex is poorly defined. It is unknown if any transcription initiation factors are incorporated into the complex after polymerase leaves the promoter region. THis proposal is a detailed characterization of the mammalian RNA polymerase II elongation complex. I have characterized a site in a eukaryotic gene that blocks transcription by RNA polymerase II and a factor (SII) that allows elongation through this site. RNA polymerase II elongation complexes will be assembled from rat liver general initiation factors which are free of elongation factors. A method has been developed to immunopurify arrested elongation complexes from in vitro transcription reactions using a monoclonal antibody against RNA. Isolated complexes resume transcript elongation when challenged with SII. The spectrum of initiation factors associated with the elongation complex will be identified. The mechanism of action and in vivo role of SII is unclear. Stable SII binding to an SII-responsive complex has not been observed. With our approach, we should be able to detect SII binding to the complex if binding has a long half-life. If the interaction is transient, we should be able to trap the SII-activated intermediate. This is an important first step in determining the biochemical basis for SII-mediated transcriptional activation. The size of the complex will be determined by nuclease footprinting experiments and electron microscopy in the presence and absence of elongation factor SII. The form of RNA polymerase II in an arrested elongation complex will be investigated. This work should facilitate our understanding of the control of transcript elongation by RNA polymerase II.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM046331-04
Application #
2183812
Study Section
Molecular Biology Study Section (MBY)
Project Start
1991-07-01
Project End
1996-06-30
Budget Start
1994-07-01
Budget End
1995-06-30
Support Year
4
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Emory University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
042250712
City
Atlanta
State
GA
Country
United States
Zip Code
30322
O'Rourke, Thomas W; Reines, Daniel (2016) Determinants of Amyloid Formation for the Yeast Termination Factor Nab3. PLoS One 11:e0150865
Loya, Travis J; Reines, Daniel (2016) Recent advances in understanding transcription termination by RNA polymerase II. F1000Res 5:
O'Rourke, Thomas W; Loya, Travis J; Head, PamelaSara E et al. (2015) Amyloid-like assembly of the low complexity domain of yeast Nab3. Prion 9:34-47
Arndt, Karen M; Reines, Daniel (2015) Termination of Transcription of Short Noncoding RNAs by RNA Polymerase II. Annu Rev Biochem 84:381-404
Loya, Travis J; O'Rourke, Thomas W; Reines, Daniel (2013) Yeast Nab3 protein contains a self-assembly domain found in human heterogeneous nuclear ribonucleoprotein-C (hnRNP-C) that is necessary for transcription termination. J Biol Chem 288:2111-7
Loya, Travis J; O'Rourke, Thomas W; Degtyareva, Natalya et al. (2013) A network of interdependent molecular interactions describes a higher order Nrd1-Nab3 complex involved in yeast transcription termination. J Biol Chem 288:34158-67
Loya, Travis J; O'Rourke, Thomas W; Reines, Daniel (2012) A genetic screen for terminator function in yeast identifies a role for a new functional domain in termination factor Nab3. Nucleic Acids Res 40:7476-91
Reines, Daniel (2012) Decapping goes nuclear. Mol Cell 46:241-2
Jenks, M Harley; O'Rourke, Thomas W; Reines, Daniel (2008) Properties of an intergenic terminator and start site switch that regulate IMD2 transcription in yeast. Mol Cell Biol 28:3883-93
Kopcewicz, Katarzyna A; O'Rourke, Thomas W; Reines, Daniel (2007) Metabolic regulation of IMD2 transcription and an unusual DNA element that generates short transcripts. Mol Cell Biol 27:2821-9

Showing the most recent 10 out of 37 publications