In bacteria, the 450 kDa RNA polymerase (RNAP) holoenzyme, comprising the evolutionarily conserved catalytic core combined with the initiation-specific ? subunit, directs transcription initiation. Bacterial transcription depends on a primary factor that is essential for viability. In addition, most bacteria contain multiple alternative ?'sthat control regulons in response to environmental cues. The principal control point of gene expression in bacteria is transcription initiation, and a major mechanism by which bacteria regulate transcription initiation is through regulation of ? activity. Dramatic insights have come from structural studies of bacterial RNAPs. Nevertheless, many challenges remain, including understanding the complex process of transcription initiation and its control by ? factors and modulators of ?-factor function. Specifically, we propose to: 1. Determine the structural basis for the recognition of the -10 element by alternative ?'s. In the last project period, we determined high-resolution crystal structures of a primary ? bound to the - 10 element sequence. How do alternative ?'s recognize and facilitate melting of their cognate -10 elements, which bear no relationship to the primary ? -10 element? To address this question, we will determine structures of alternative ?'s bound to their -10 element sequence. 2. Structurally characterize the role of ?N region I in promoter recognition. The ?N family of ? factors (also called ?54) is evolutionarily unrelated to the ?70 family and functions through a distinct mechanism. In the last funding period, we determined crystal structures of ?N/promoter DNA complexes, but these structures lack the N-terminal ?N region I, which controls promoter melting. We will determine crystal structures of full-length ?N bound to promoter DNA constructs. 3. Determine the structure of a Sau phage G1 ORF67/Sau ?A4/DNA complex. In the last funding period, our studies revealed that ORF67 targets transcription in a promoter-specific manner by disrupting RNAP ?-subunit/UP-element interactions, but the molecular basis for how this is achieved was not clear from our ORF67/?A4 structures. To understand the molecular details of ORF67 function, we will determine the crystal structure of an ORF67/?A4/promoter DNA ternary complex. 4. Determine the structure of a new family of anti-?: Streptomyces venezuelae ?BldN/RsbN. An ECF ?/anti-? pair, ?BldN/RsbN, plays a critical role in the cellular differentiation of Streptomyces into aerial hyphae and spores. The biological cue sensed by RsbN to release ?BldN is unknown, and sequence analysis also indicates RsbN represent a new class of anti-?. A crystal structure of the ?BldN/RsbN complex will help address these questions.

Public Health Relevance

We focus on highly characterized bacterial RNA polymerases, which have a high degree of conservation of structure and function from bacteria to man. The bacterial RNA polymerase is a proven target for antimicrobials, such as rifampicin (or its derivatives), widely used in combination therapy to treat tuberculosis, but 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 #
5R01GM053759-19
Application #
8643236
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Sledjeski, Darren D
Project Start
1996-03-01
Project End
2017-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
19
Fiscal Year
2014
Total Cost
$533,193
Indirect Cost
$218,625
Name
Rockefeller University
Department
Physiology
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
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
Feklistov, Andrey; Darst, Seth A (2013) Crystallographic analysis of an RNA polymerase ýý-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
Feklistov, Andrey; Darst, Seth A (2011) Structural basis for promoter-10 element recognition by the bacterial RNA polymerase ? subunit. Cell 147:1257-69
Twist, Kelly-Anne F; Campbell, Elizabeth A; Deighan, Padraig et al. (2011) Crystal structure of the bacteriophage T4 late-transcription coactivator gp33 with the ?-subunit flap domain of Escherichia coli RNA polymerase. Proc Natl Acad Sci U S A 108:19961-6
Devi, Pukhrambam Grihanjali; Campbell, Elizabeth A; Darst, Seth A et al. (2010) Utilization of variably spaced promoter-like elements by the bacterial RNA polymerase holoenzyme during early elongation. Mol Microbiol 75:607-22
Opalka, Natacha; Brown, Jesse; Lane, William J et al. (2010) Complete structural model of Escherichia coli RNA polymerase from a hybrid approach. PLoS Biol 8:
Lane, William J; Darst, Seth A (2010) Molecular evolution of multisubunit RNA polymerases: structural analysis. J Mol Biol 395:686-704
Lane, William J; Darst, Seth A (2010) Molecular evolution of multisubunit RNA polymerases: sequence analysis. J Mol Biol 395:671-85
Feklistov, Andrey; Darst, Seth A (2009) Promoter recognition by bacterial alternative sigma factors: the price of high selectivity? Genes Dev 23:2371-5

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