Timely and accurate regulation of gene expression is required for proper growth, development, and response to environmental stimuli. A complete understanding of the mechanisms employed to regulate gene expression is necessary to combat the aberrant regulation that underlies many human developmental conditions and ailments, including cancer. The long-term goals of this proposal are to determine the mechanics of RNA synthesis by DNA-dependent multi-subunit RNA polymerases (RNAPs), the regulation imposed on RNAP by conserved protein factors and select template sequences, and to specifically characterize the available mechanisms to halt RNA synthesis and terminate transcription. The proposed experiments take advantage of both an in vivo and in vitro established archaeal transcription system. Archaea offer the advantages of far less complexity but homology in many features of human molecular biology, specifically conservation of RNAP structure and function. We will select, generate, purify and characterize variant RNAPs with modified transcription termination phenotypes. We will further characterize factor-dependent termination, polarity, and the role of such regulation in Archaea. The results so obtained will determine the structures and sequences that support transcription elongation complex stability, describe the mechanics of termination and allow comparisons of termination mechanisms in each Domain, and open a new field of study, archaeal factor-dependent termination.

Public Health Relevance

A complete understanding of the mechanisms employed to regulate gene expression is necessary to combat the aberrant regulation that underlies many human developmental conditions and ailments, including cancer. The long-term goals of this proposal are to determine the mechanics of RNA synthesis by DNA-dependent multi-subunit RNA polymerases (RNAPs), the regulation imposed on RNAP by conserved protein factors and select template sequences, and to specifically characterize the available mechanisms to halt RNA synthesis and terminate transcription. The results so obtained will determine the structures and sequences that support transcription elongation complex stability, describe the mechanisms of termination and allow comparisons of termination mechanisms in each Domain, and open a new field of study, archaeal factor-dependent termination.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM100329-03
Application #
8698877
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Sledjeski, Darren D
Project Start
2012-02-01
Project End
2016-12-31
Budget Start
2013-07-01
Budget End
2013-12-31
Support Year
3
Fiscal Year
2013
Total Cost
$246,872
Indirect Cost
$75,872
Name
Colorado State University-Fort Collins
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
785979618
City
Fort Collins
State
CO
Country
United States
Zip Code
80523
Jun, Sung-Hoon; Hirata, Akira; Kanai, Tamotsu et al. (2014) The X-ray crystal structure of the euryarchaeal RNA polymerase in an open-clamp configuration. Nat Commun 5:5132
Li, Zhuo; Huang, Richard Y-C; Yopp, Daniel C et al. (2014) A novel mechanism for regulating the activity of proliferating cell nuclear antigen by a small protein. Nucleic Acids Res 42:5776-89
Jäger, Dominik; Förstner, Konrad U; Sharma, Cynthia M et al. (2014) Primary transcriptome map of the hyperthermophilic archaeon Thermococcus kodakarensis. BMC Genomics 15:684