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Chemistry & Chemical Biology / New Brunswick |
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Richard H. Ebright Professor A.B., 1981, Ph.D. 1987, Harvard Harvard Junior Fellow 1984-1987 |
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Research
Summary
Transcription initiation, the first step in gene expression, is the step at which most regulation of gene expression occurs. Richard Ebrights lab seeks to understand the structure, function, and regulation of transcription initiation complexes and to develop gene-specific inhibitors of transcription initiation as potential therapeutic agents.Structure of Transcription Initiation Complexes
Transcription initiation in bacteria requires RNA polymerase and the initiation factor s. The bacterial transcription initiation complex contains six polypeptides (five in RNA polymerase, one in s) 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, the intact complexes have proved refractory to crystallographic structure determination. Therefore, efforts to understand the arrangement of polypeptides within the intact complexes rely heavily on biophysical data defining distances within the complexes and on biochemical and genetic data defining contacts within the 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 Initiation 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 and nanomanipulation for single-molecule, millisecond- to second-scale analysis of transitions within transcription complexes.
Regulation of Transcription Initiation Complexes
Crystallographic structure of a transcriptional activator (catabolite activator protein, CAP; cyan) in complex with its target in the transcriptional machinery (RNA polymerase a-subunit C-terminal domain, aCTD; green) and DNA (red). There are no large-scale conformational changes in the activator and target, and the interface between the activator and target is small (residues highlighted in navy and yellow)—consistent with the proposal that transcriptional activation involves a simple "recruitment" mechanism.See Benoff, B., Yang, H., Lawson, C.L., Parkinson, G., Liu, J., Blatter, E., Ebright, Y.W., Berman, H.M., and Ebright, R.H. 2002. Science 297:1562–1566.
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.
With the objective of providing 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 CC(41.5) promoter by CAP. These model systems each involve only a single activator molecule and a single activator DNA site, and, thus, 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 a-subunit C-terminal domain that facilitates closed-complex formation. Activation at CC(41.5) involves this same interaction and also an interaction between CAP and the RNA polymerase a-subunit N-terminal domain that facilitates 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 the FRET, photocrosslinking, and single-moleculeoptical-microscopy 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.
Low-Molecular-Weight Inhibitors of Transcription Initiation
Inhibition of the interaction of an activator with DNA or with the general transcription machinery results in selectively reduced expression of the gene(s) regulated by the activator. In principle, development of low-molecular-weight inhibitors of interactions of specific activators with DNA or with the general transcription machinery could provide highly selective regulators of gene expression and thus highly selective therapeutic agents (e.g., antimicrobial agents based on inactivation of a critical microbial activator).We are using genetic, biochemical, and crystallographic approaches to define mechanisms of action of known inhibitors of transcription, and combinatorial chemistry and peptidomimetic chemistry approaches to seek novel inhibitors of transcription. In addition, we are developing and testing methods for optically encoded combinatorial chemistry.
Awards & Honors
Johnson & Johnson Discovery Research Fellow 1990-1992
Walter J. Johnson Prize 1995
American Society for Biochemistry and Molecular Biology/Schering-Plough Research Achievement Award 1995
American Academy of Microbiology 1996
Howard Hughes Medical Institute, 1997
Representative Publications
Chen, Y., Ebright, Y., and Ebright, R. (1994) Identification of the target of a transcription activator protein by protein-protein photocrosslinking. Science 265, 90-92.
Pendergrast, P.S., Ebright, Y., and Ebright, R. (1994) High-specificity DNA cleavage agent: design and application to kilobase and megabase DNA substrates. Science 265, 959-961.
Blatter, E., Ross, W., Tang, H., Gourse, R., and Ebright, R. (1994) Domain organization of RNA polymerase a subunit: C-terminal 85 amino acids constitute a domain capable of dimerization and DNA binding. Cell 78, 889-896.
Busby, S. and Ebright, R. (1994) Promoter structure, promoter recognition, and transcription activation in prokaryotes. Cell 79, 743-746.
Tang, H., Sun, X., Reinberg, D., and Ebright, R. (1996) Protein-protein interactions in eukaryotic transcription intiation: structure of the pre-initiation complex. Proc. Natl. Acad. Sci. USA 93, 1119-1124.
Pellegrini, M. and Ebright, R. (1996) Artificial DNA binding peptides. J. Amer. Chem. Soc. 118, 5831-5835.
Niu, W., Kim, Y., Tau, G., Heyduk, and Ebright, R. (1996) Transcription activation at Class II CAP-dependent promoters: two interactions between CAP and RNA polymerase. Cell 87, 1123-1134.
Miller, A., Wood, D., Ebright, R., and Rothman-Denes, L. (1997) RNA polymerase ß: a target for DNA-binding-independent activation. Science 275, 1655-1657.
Kim, T.-K., Lagrange, T., Reinberg, D., and Ebright, R. (1997) Trajectory of DNA in the RNA polymerase II transcription preinitiation complex Proc. Natl. Acad. Sci. USA 94, 12268-12273.
Lagrange, T., Kapanidis, A., Tang, H., Reinberg, D., and Ebright, R. (1998) New core promoter element in RNA polymerase II-dependent transcription: sequence-specific DNA binding by transcription factor IIB. Genes & Development, 12, 34-44.
Estrem, S., Ross, W., Gaal., Chen, Z.W.S., Niu, W., Ebright, R., and Gourse, R. (1999) Bacterial promoter architecture: subsite structure of UP elements and interactions with the carboxyl-terminal domain of RNA polymerase alpha subunit. Genes & Development 13, 2134-2147.
Tan, Q., Linask, K.L., Ebright, R. and Woychik, N. (2000) Activation mutants in yeast RNA polymerase subunit RPB3 provide evidence for a structurally conserved surface required for activation in eukaryotes and bacteria. Genes & Development 14, 339-348.
Kim, T.-K., Ebright, R., and Reinberg, D. (2000) Mechanism of ATP-dependent promoter melting by transcription factor IIH. Science 288, 1418-1421.
Naryshkin, N., Revyakin, A., Kim, Y., Mekler, V., and Ebright, R. (2000) Structural organization of the RNA polymerase-promoter open complex. Cell 101, 601-611.
Minakhin, L., Bhagat, S. Brunning, A., Campbell, E., Darst, S., Ebright, R. and Severinov, K. (2001) Bacterial RNA polymerase subunit omega and eukaryotic RNA polymerase subunit RPB6 are sequence, structural, and functional homologs and promote RNA polymerase assembly Proc. Natl. Acad. Sci. USA 98, 892-897.
Mukhopadhyay, J., Kapanidis, A., Mekler, V., Kortkhonjia, E., Ebright, Y., and Ebright, R. (2001) Translocation of sigma70 with RNA polymerase during transcription: fluorescence resonance energy transfer assay for movement relative to DNA. Cell 106, 453-463.
Kapanidis, A., Ebright, Y., and Ebright, R. (2001) Site-specific incorporation of fluorescent probes into protein: hexahistidine-tag-mediated fluorescent labeling using (Ni2+:nitrilotriacetic acid)n-fluorochrome conjugates. J. Amer. Chem. Soc. 123, 12123-12125.
Mekler, V., Kortkhonjia, E., Mukhopadhyay, J., Knight, J., Revyakin, A., Kapanidis, A., Niu, W., Ebright, Y., Levy, R., and Ebright, R. (2002) Structural organization of RNA polymerase holoenzyme and the RNA polymerase-promoter open complex. Cell 108, 599-614.
Benoff, B., Yang, H., Lawson, C., Parkinson, G., Liu, J., Blatter, E., Ebright, Y., Berman, H., and Ebright, R. (2002) Structural basis of transcription activation: structure of the CAP-αCTD-DNA complex. Science 297, 1562-1566.
Lloyd, G., Niu, W., Trebbutt, J., Ebright, R., and Busby, S. (2002) Requirement for two copies of RNA polymerase α subunit C-terminal domain for synergistic transcription activation at complex bacterial promoters. Genes & Development 16, 2557-2565.
Mekler, V., Kortkhonjia, E., Mukhopadhyay, J., Knight, J., Revyakin, A., Kapanidis, A., Niu, W., Ebright, Y., Levy, R., and Ebright, R. (2002) Structural organization of RNA polymerase holoenzyme and the RNA polymerase-promoter open complex. Cell 108, 599-614.
Benoff, B., Yang, H., Lawson, C., Parkinson, G., Liu, J., Blatter, E., Ebright, Y., Berman, H., and Ebright, R. (2002) Structural basis of transcription activation: structure of the CAP-aCTD-DNA complex. Science 297, 1562-1566.
Chen, H., Tang, H., and Ebright, R.H. (2003) Functional interaction between RNA polymerase a subunit C-terminal domain and s70 in UP-element- and activator-dependent transcription. Mol. Cell 11, 1621-1623.
Bayro, M., Mukhopadhyay, J., Swapna, G.V.T., Huang, J., Ma, L.-C., Sineva, E., Dawson, P., Montelione, G., and Ebright, R. (2003) Structure of antibacterial peptide microcin J25: a 21-residue lariat protoknot. J. Amer. Chem. Soc. 125, 12382-12383.
Revyakin, A., Ebright, R., and Strick, T. (2004) Promoter unwinding and promoter clearance by RNA polymerase: Detection by single-molecule DNA nanomanipulation. Proc. Natl. Acad. Sci. USA 101, 4776-4780.
Nickels, B., Mukhopadhyay, J., Garrity, S., Ebright, R., and Hochschild, A. (2004) s70 mediates a promoter-proximal pause at the lac promoter. Nature Structl. Mol. Biol. 11, 544-550.
Mukhopadhyay, J., Sineva, E., Knight, J., Levy, R., and Ebright, R. (2004) Antibacterial peptide microcin J25 (MccJ25) inhibits transcription by binding within and obstructing the RNA polymerase secondary channel. Mol. Cell. 14, 739-751.
Knight, J., Mekler, V., Mukhopadhyay, J., Ebright, R., Levy, R. (2005) Distance-restrained docking of rifampicin and rifamycin SV to RNA polymerase using systematic FRET measurements: developing benchmarks of model quality and reliability. Biophys J. 88, 925-938.
Revyakin, A., Ebright, R., and Strick, T. (2005) Single-molecule DNA nanomanipulation: improved resolution through use of shorter DNA fragments. Nature Meths. 2, 127-138.