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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Special Emphasis Panel (ZRG1-GGG-N (08))
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Hagan, Ann A
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Rockefeller University
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New York
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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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