In bacteria, the 450 kDa RNA polymerase (RNAP) holoenzyme, comprising the evolutionarily conserved catalytic core (subunit composition ?2??'?) combined with the initiation-specific ? subunit, directs transcription initiation. Bacterial transcription depends on a primary ? factor that is essential for viability, as well as alternative ?'s that control specific regulons. A major mechanism to control transcription initiation is through regulation of ? activity. Dramatic insights have come from structural studies of ?'s and holoenzymes. Nevertheless, many challenges remain. In this competing continuation, we propose studies to further our understanding of ? factor structure and function, and interactions with accessory factors. Specifically, we propose to: 1. Determine the structural basis for ? interactions with the -10 element in the initiation of promoter melting. We will determine crystal structures of complexes between ? and in vitro- selected DNA aptamers that mimic ?/-10 element interactions critical for promoter melting. 2. Map the position of E. coli ?701.1 on the RNAP holoenzyme during different stages of promoter open complex formation. A protein-protein crosslinking/mapping approach (guided by our new structure of ?1.1) will be used. The crosslinking-based data will be combined with existing FRET-based distance constraints to generate structural models of ?701.1 in the context of the RNAP holoenzyme in holoenzyme alone, as well as in closed and open promoter complexes. 3. Structurally and functionally characterize ?/anti-? complexes: Staphylococcal aureus phage G1 ORF67. S. aureus phage G1 ORF67 binds to S. aureus ?A domain 4 and is a potent inhibitor of S. aureus transcription. We will determine crystal structures of complexes between phage G1 ORF67 and S. aureus ?A domain 4, and functionally characterize the mechanism of G1 ORF67 inhibition of S. aureus transcription. 4. Determine the structural basis for ?N interactions with its promoter DNA. We will determine the crystal structure of a complex between Aquifex aeolicus ?N and promoter DNA.

Public Health Relevance

Transcription is the major control point of gene expression and RNA polymerase is the central enzyme of transcription. Our long term goal is to understand the mechanism of transcription and its regulation. We focus on highly characterized prokaryotic RNA polymerases, which have a high degree of conservation of structure and function from bacteria to man. The ? factors are the key regulators of bacterial transcription initiation. Defective transcription regulation underlies many disorders. For example, many oncoproteins are transcriptional activator proteins. Moreover, the bacterial RNA polymerase is a proven target for antimicrobials, such as rifampicin (or its derivatives), widely used in combination therapy to treat tuberculosis. Bacterial strains resistant to rifampicin arise with appreciable frequency, compromising treatment. Insights into the mechanism of bacterial transcription can lead to new avenues for the development of antimicrobials.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM053759-17
Application #
8249858
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Sledjeski, Darren D
Project Start
1996-03-01
Project End
2013-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
17
Fiscal Year
2012
Total Cost
$524,654
Indirect Cost
$214,430
Name
Rockefeller University
Department
Physiology
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
Bae, Brian; Nayak, Dhananjaya; Ray, Ananya et al. (2015) CBR antimicrobials inhibit RNA polymerase via at least two bridge-helix cap-mediated effects on nucleotide addition. Proc Natl Acad Sci U S A 112:E4178-87
Bae, Brian; Feklistov, Andrey; Lass-Napiorkowska, Agnieszka et al. (2015) Structure of a bacterial RNA polymerase holoenzyme open promoter complex. Elife 4:
Hubin, Elizabeth A; Tabib-Salazar, Aline; Humphrey, Laurence J et al. (2015) Structural, functional, and genetic analyses of the actinobacterial transcription factor RbpA. Proc Natl Acad Sci U S A 112:7171-6
Feklistov, Andrey; Darst, Seth A (2013) Crystallographic analysis of an RNA polymerase σ-subunit fragment complexed with -10 promoter element ssDNA: quadruplex formation as a possible tool for engineering crystal contacts in protein-ssDNA complexes. Acta Crystallogr Sect F Struct Biol Cryst Commun 69:950-5
Osmundson, Joseph; Darst, Seth A (2013) Biochemical insights into the function of phage G1 gp67 in Staphylococcus aureus. Bacteriophage 3:e24767
Feklistov, Andrey (2013) RNA polymerase: in search of promoters. Ann N Y Acad Sci 1293:25-32
Montero-Diez, Cristina; Deighan, Padraig; Osmundson, Joseph et al. (2013) Phage-encoded inhibitor of Staphylococcus aureus transcription exerts context-dependent effects on promoter function in a modified Escherichia coli-based transcription system. J Bacteriol 195:3621-8
Bae, Brian; Davis, Elizabeth; Brown, Daniel et al. (2013) Phage T7 Gp2 inhibition of Escherichia coli RNA polymerase involves misappropriation of σ70 domain 1.1. Proc Natl Acad Sci U S A 110:19772-7
Osmundson, Joseph; Dewell, Scott; Darst, Seth A (2013) RNA-Seq reveals differential gene expression in Staphylococcus aureus with single-nucleotide resolution. PLoS One 8:e76572
Osmundson, Joseph; Montero-Diez, Cristina; Westblade, Lars F et al. (2012) Promoter-specific transcription inhibition in Staphylococcus aureus by a phage protein. Cell 151:1005-16

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