Structures of Transcription Complexes
Transcription initiation in bacteria requires RNA polymerase (RNAP) and the transcription initiation factor σ. The bacterial transcription initiation complex contains six polypeptides (five in RNAP, one in σ) and promoter DNA, and has a molecular mass of 0.5 MDa.
Understanding bacterial transcription initiation will require understanding the structures of polypeptides in bacterial transcription initiation complexes and the arrangements of these polypeptides relative to each other and relative to promoter DNA.
Crystal structures have been determined for individual components of bacterial transcription initiation complexes. However, intact, fully assembled, transcription initiation complexes have proved refractory to crystallographic structure determination. Therefore, efforts to understand the arrangement of polypeptides within intact transcription initiation complexes rely heavily on biophysical data defining distances within complexes and on biochemical and genetic data defining contacts within complexes.
We are analyzing distances, protein-protein contacts, and protein-DNA contacts within bacterial transcription initiation complexes. We are using ensemble fluorescence resonance energy transfer (FRET) to define distances between pairs of site-specifically incorporated fluorescent probes, photocrosslinking to define polypeptides near site-specifically incorporated photocrosslinking probes, and protein footprinting and residue scanning to define residues involved in contacts. In support of these activities, we are developing procedures to incorporate fluorescent probes, photocrosslinking probes, and other biophysical and biochemical probes at specific sites within large multisubunit complexes. Finally, we are developing and using automated constrained docking algorithms to integrate structural, biophysical, biochemical, and genetic data in order to construct models for structures of complexes.
Mechanisms of Transcription Complexes
Transcription complexes are molecular machines that carry out complex, multistep reactions in transcription initiation and elongation:
(1) RNAP binds to promoter DNA, to yield an RNAP-promoter closed complex.
(2) RNAP unwinds ~14 base pairs of promoter DNA surrounding the transcription start site, rendering accessible the genetic information in the template strand of DNA, and yielding an RNAP-promoter open complex.
(3) RNAP begins synthesis of RNA as an RNAP-promoter initial transcribing complex. During initial transcription, RNAP uses a "scrunching" mechanism, in which RNAP remains stationary on promoter DNA and unwinds and pulls downstream DNA into itself and past its active center in each nucleotide-addition cycle, resulting in generation of a stressed intermediate.
(4) When RNAP synthesizes an RNA product of a threshold length (~9–11 nucleotides at most promoters; ~14–20 nucleotides at some promoters), RNAP breaks its interactions with promoter DNA, breaks at least some of its interactions with σ, escapes the promoter, and begins transcription elongation as a transcription elongation complex. Energy stored in the stressed intermediate generated by scrunching during initial transcription is used to drive breakage of interactions with promoter DNA and interactions with σ during promoter escape. During transcription elongation, RNAP uses a "stepping" mechanism, in which RNAP translocates relative to DNA in each nucleotide-addition step.
Each nucleotide-addition cycle during initial transcription and transcription elongation can be further subdivided into four substeps: (1) translocation of the RNAP active center relative to DNA (by scrunching in initial transcription; by stepping in transcription elongation), (2) binding of the incoming nucleotide, (3) formation of the phosphodiester bond, and (4) release of pyrophosphate. Crystal structures have been reported for transcription elongation complexes without incoming nucleotides and for transcription elongation complexes with incoming nucleotides. Based on these crystal structures, it has been proposed that each nucleotide-addition cycle is coupled to an RNAP active-center conformational cycle, involving closing of the RNAP active center upon binding of the incoming nucleotide, followed by opening of the RNAP active center upon formation of the phosphodiester bond. According to this proposal, the closing and opening of the RNAP active center is mediated by the folding and the unfolding of an RNAP active-center structural element, the "trigger loop."
To understand transcription initiation, transcription elongation, and transcriptional regulation, it will be necessary to leverage, and to go beyond, the available crystallographic structural information, in order to define the structural transitions in RNAP and nucleic acid in each reaction, to define the kinetics of each reaction, and to define mechanisms of regulation of each reaction.
We are using the FRET and photocrosslinking methods to define distances and contacts within trapped intermediates in transcription initiation and transcription elongation. In addition, we are using FRET with stopped-flow rapid mixing, and photocrosslinking with quenched-flow rapid mixing and laser flash photolysis, to monitor kinetics of structural transitions. Finally, and most importantly, we are using single-molecule FRET, single-molecule DNA nanomanipulation, and combined single-molecule FRET and single-molecule DNA nanomanipulation, to carry out single-molecule, millisecond-to-second timescale analysis of structural transitions.
Regulation of Transcription Complexes
The activities of bacterial transcription initiation complexes are regulated in response to environmental, cell-type, and developmental signals. In most cases, regulation is mediated by factors that bind to specific DNA sites in or near a promoter and inhibit (repressors) or stimulate (activators) one or more of the steps on the transcription initiation pathway.
To provide the first complete structural and mechanistic descriptions of activation, we study two of the simplest examples of activation in bacteria: (1) activation of the lac promoter by catabolite activator protein (CAP) and (2) activation of the gal promoter by CAP. These model systems each involve only a single activator molecule and a single activator DNA site and, as such, are more tractable than typical examples of activation in bacteria and substantially more tractable than typical examples of activation in eukaryotes (which can involve tens of activator molecules and activator DNA sites).
We have established that activation at lac involves an interaction between CAP and the RNAP α-subunit C-terminal domain that facilitates closed-complex formation. Activation at gal involves this same interaction and also interactions between CAP and the RNAP α-subunit N-terminal domain, and between CAP and σ, that facilitate isomerization of closed complex to open complex.
Together with collaborators, we are using electron microscopy, x-ray crystallography, and nuclear magnetic resonance (NMR) spectroscopy to determine the structures of the interfaces between CAP and its targets on RNAP. In addition, we are using FRET, photocrosslinking, and single-molecule FRET and single-molecule DNA nanomanipulation methods to define when each CAP-RNAP interaction is made as RNAP enters the promoter and when each interaction is broken as RNAP leaves the promoter.
Small-Molecule Inhibitors of Bacterial Transcription
Bacterial RNAP is a proven target for broad-spectrum antibacterial therapy. The suitability of bacterial RNAP as a target for broad-spectrum antibacterial therapy follows from the fact that bacterial RNAP is an essential enzyme (permitting efficacy), the fact that bacterial RNAP-subunit sequences are highly conserved (providing a basis for broad-spectrum activity), and the fact that bacterial RNAP-subunit sequences are not highly conserved in human RNAPI, RNAPII, and RNAPIII (providing a basis for therapeutic selectivity).
The rifamycin antibacterial agents—notably rifampin, rifapentine, and rifabutin—bind to and inhibit bacterial RNAP. The rifamycins bind to a site on bacterial RNAP adjacent to the RNAP active center and prevent extension of RNA chains beyond a length of 2–3 nucleotides. The rifamycins are in current clinical use in treatment of Gram-positive and Gram-negative bacterial infections. The rifamycins are of particular importance in treatment of tuberculosis; the rifamycins are first-line antituberculosis agents and are among the only antituberculosis agents able to clear infection and prevent relapse. The clinical utility of the rifamycin antibacterial agents is threatened by the existence of bacterial strains resistant to rifamycins. Resistance to rifamycins typically involves substitution of residues in or adjacent to the rifamycin-binding site on bacterial RNAP—i.e., substitutions that directly interfere with rifamycin binding.
In view of the public health threat posed by drug-resistant and multidrug-resistant bacterial infections, there is an urgent need for new classes of broad-spectrum antibacterial agents that (1) target bacterial RNAP (and thus have the same biochemical effects as rifamycins), but that (2) target sites within bacterial RNAP that do not overlap the rifamycin-binding site (and thus do not show cross-resistance with rifamycins).
We have identified new drug targets within the structure of bacterial RNAP. Each of these new targets can serve as a potential binding site for compounds that inhibit bacterial RNAP and thereby kill bacteria. Each of these new targets is present in most or all bacterial species, and thus compounds that bind to these new targets are active against a broad spectrum of bacterial species. Each of these new targets is different from targets of current antibiotics, and thus compounds that bind to these new targets are not cross-resistant with current antibiotics. For each of these new targets, we have identified at least one lead compound that binds to the target, and we have synthesized analogs of the lead compound comprising optimized lead compounds. Several of the lead compounds and optimized lead compounds are promising: they exhibit potent activity against a broad spectrum of bacterial pathogens (including Mycobacterium tuberculosis, Staphylococcus aureus, Enterococcus faecalis, Bacillus anthracis, and Yersinia pestis) and exhibit no cross-resistance with current antibiotics.
In support of this work, we are identifying new small-molecule inhibitors of bacterial RNAP by analysis of microbial and plant natural products, by high-throughput screening, and by virtual screening. We are also using genetic, biochemical, biophysical, and crystallographic approaches to define the mechanism of action of each known, and each newly identified, small-molecule inhibitor of bacterial RNAP, and we are using microbiological approaches to define antibacterial efficacies, resistance spectra, and spontaneous resistance frequencies of known and new small-molecule inhibitors of bacterial RNAP.
We seek to address the following objectives: to develop new classes of antituberculosis agents and broad-spectrum antibacterial agents, to develop antibacterial agents effective against pathogens resistant to current antibiotics, to develop antibacterial agents effective against pathogens of high relevance to public health, and to develop antibacterial agents effective against pathogens of high relevance to biodefense.