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 research, we initially proposed studies to further our understanding of ? factor structure and function, and interactions with accessory factors. Specifically, we proposed to: 1. Determine the structural basis for ? interactions with the -10 element in the initiation of promoter melting. 2. Determine the structural basis for ?N interactions with its promoter DNA. 3. Structurally and functionally characterize bacteriophage transcriptional regulators. While significant progress has been achieved for all of these Aims, this competetive revision focuses on Aim 1, where results have led to new hypotheses, the testing of which expands the scope of the original Aim. The key step in bacterial promoter opening is recognition of the -10 promoter element by the RNAP ? subunit. We have determined high-resolution crystal structures of ? domain 2 bound to single-stranded DNA bearing -10 element sequences. Structural analysis leads to a model in which the -10 element sequence is not recognized in its double-stranded form, only in single-stranded form after base flipping through thermal breathing of the double-helix. The ?2-mediated capture of otherwise transiently flipped -10 element non-template-strand bases provides the crucial stability to the initial strand separated state. This model is contrary to current thinking in the field. Here we propose to test this model for the mechanism of the initiation of promoter melting through detailed biochemical and structural studies using synthetic DNA oligonucleotides containing various nucleotide analogs. These studies will specifically address whether the RNAP holoenzyme interacts with the double-stranded -10 element in a sequence-specific manner. Depending on the results, the studies may also provide support for the proposal that initial exposure of the DNA bases occurs through a thermal breathing mechanism.

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 #
3R01GM053759-16S1
Application #
8237581
Study Section
Special Emphasis Panel (ZRG1-GGG-N (08))
Program Officer
Hagan, Ann A
Project Start
1996-03-01
Project End
2013-04-30
Budget Start
2012-01-01
Budget End
2012-04-30
Support Year
16
Fiscal Year
2012
Total Cost
$114,713
Indirect Cost
$27,213
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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