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Structure, Function, and Regulation of Transcription Complexes
Summary: Transcription, the first step in gene expression, is the step at which most regulation of gene expression occurs. Richard Ebright's lab seeks to understand the structure, function, and regulation of transcription complexes and to identify and characterize inhibitors of bacterial transcription for use as potential antibacterial agents.
Structure of Transcription Complexes
Transcription initiation in bacteria requires RNA polymerase and the initiation factor σ. The bacterial transcription initiation complex contains six polypeptides (five in RNA polymerase, one in σ) and promoter DNA, and has a molecular mass of 0.5 MDa.
Transcription initiation at a eukaryotic protein-encoding gene involves RNA polymerase II, SRB/Med components, and up to six general transcription factors: IIA, IIB, IID, IIE, IIF, and IIH. The fully assembled eukaryotic transcription initiation complex contains more than 50 polypeptides (12 in RNA polymerase II, at least 15 in SRB/Med, and at least 26 in general transcription factors) and promoter DNA, and has a molecular mass in excess of 3 MDa.
Understanding transcription initiation in bacteria and eukaryotes will require understanding the structures of the polypeptides in the respective transcription initiation complexes and the arrangement of these polypeptides relative to each other and relative to promoter DNA.
Crystallographic structures have been determined for several components of the bacterial and eukaryotic transcription initiation complexes. However, intact 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 the bacterial and eukaryotic transcription initiation complexes. We are using 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 addition, we are using binding-site selection to define new promoter DNA sequence elements recognized by polypeptides and polypeptide fragments. Finally, we are developing and using automated constrained docking algorithms to integrate structural, biophysical, biochemical, and genetic data in order to construct models for the structures of complexes.
Function of Transcription Complexes
The bacterial and eukaryotic transcription initiation complexes are molecular machines that carry out complex, multistep reactions. The transcription initiation pathway involves (1) binding of RNA polymerase and initiation factor(s) to promoter DNA to form a "closed complex" with duplex DNA; (2) isomerization through several intermediates to form an "open complex" with an ~14-nucleotide (nt) region of melted, single-stranded DNA surrounding the transcription start; (3) abortive cycles of synthesis and release of 2- to 8-nt RNA oligomers as an "initial transcribing complex"; and (4) upon synthesis of a 9-nt RNA oligomer, isomerization to break protein-DNA interactions between RNA polymerase and the promoter and to break, or weaken, protein-protein interactions between RNA polymerase and initiation factor(s), resulting in an "elongation complex" that processively translocates along DNA and extends the RNA product.
Each step in this pathway appears to involve conformational changes in both RNA polymerase and promoter DNA. Understanding transcription initiation will require defining the structure of the complex at each step, defining the conformational transitions, and defining the kinetics of the transitions.
We are addressing these issues in studies of the smaller, and thus more experimentally tractable, bacterial transcription complex. We are using the FRET and photocrosslinking methods of the preceding section to define distances and contacts within trapped intermediates (e.g., closed complexes trapped at 4°C, intermediate complexes trapped at 15°C, open complexes trapped at 37°C in the absence of NTPs, initial transcribing complexes trapped at 37°C in the presence of specific subsets of NTPs). 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 transitions. Finally, we are using single-molecule optical microscopy, single-molecule DNA nanomanipulation, and combined single-molecule optical microscopy and single-molecule DNA nanomanipulation, for single-molecule, millisecond- to second-scale analysis of transitions within transcription complexes.
Regulation of Transcription Complexes
The activities of the bacterial and eukaryotic 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 RNA polymerase α-subunit C-terminal domain that facilitates closed-complex formation. Activation at gal involves this same interaction and also interactions between CAP and the RNA polymerase α-subunit N-terminal domain, and between CAP and σ, that facilitate isomerization of closed complex to open complex.
We are using x-ray crystallography to determine the structures of the interfaces between CAP and its targets on RNA polymerase, and we are using FRET, photocrosslinking, and single-molecule–optical-microscopy and single-molecule–DNA-nanomanipulation methods to define when each CAP-RNA polymerase interaction is made as RNA polymerase enters the promoter and when each interaction is broken as RNA polymerase leaves the promoter.
Inhibitors of Bacterial Transcription
Bacterial transcription is a proven target for antibacterial therapy. The rifamycin antibacterial agents—notably rifampicin, rifapentine, and rifabutin—bind to and inhibit bacterial transcription complexes. Due to the public health threat posed by drug-resistant and multidrug-resistant bacterial infection, there is an urgent need for novel classes of antibacterial agents that target bacterial transcription complexes (and thus have the same biochemical effects as rifamycin antibacterial agents) but that target different, non-overlapping structural elements within bacterial transcription complexes (and thus do not show cross-resistance with rifamycin antibacterial agents).
We are identifying and characterizing small molecules that target specific structural elements within bacterial transcription complexes and/or that inhibit specific reaction steps of these complexes. We are using genetic, biochemical, spectroscopic, and crystallographic approaches to define the mechanism of action of each known inhibitor of bacterial transcription; peptidomimetic-chemistry approaches to develop improved inhibitors of bacterial transcription; and combinatorial-chemistry, high-throughput screening, and structure-based screening approaches to identify novel inhibitors of transcription. In addition, we are developing and testing methods for optically encoded combinatorial chemistry.
Grants from the National Institutes of Health provided partial support for the projects described above.
Last updated: April 26, 2006
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