Transcription--synthesis of an RNA copy of genetic information in DNA--is the first step in gene expression and is the step at which most regulation of gene expression occurs. The lab seeks to understand structures, mechanisms, and regulation of bacterial transcription complexes and to identify, characterize, and develop small-molecule inhibitors of bacterial transcription for application as antituberculosis agents and broad-spectrum antibacterial agents.
190 Frelinghuysen Road
Piscataway, NJ 08854
Structural basis of transcription inhibition by fidaxomicin (lipiarmycin A3)
Lin W, Das K, Degen D, Mazumder A, Duchi D, Wang D, Ebright YW, Ebright RY, Sineva E, Gigliotti M, Srivastava A, Mandal S, Jiang Y, Liu Y, Yin R, Zhang Z, Eng ET, Thomas D, Donadio S, Zhang H, Zhang C, Kapanidis AN, Ebright RH
Molecular Cell 7:60-71, 2018
Antibacterial nucleoside-analog inhibitor of bacterial RNA polymerase
Maffioli S, Zhang Y, Degen D, Carzaniga T, Del Gatto G, Serina S, Monciardini P, Mazzetti C, Guglierame P, Candiani G, Chiriac AI, Facchetti G, Kaltofen P, Sahl HG, Dehò G, Donadio S, Ebright RH
Cell 169:1240-1248, 2017
Structural basis of Mycobacterium tuberculosis transcription and transcription inhibition
Lin W, Mandal S, Degen D, Liu Y, Ebright YW, Li S, Feng Y, Zhang Y, Mandal S, Jiang Y, Liu S, Gigliotti M, Talaue M, Connell N, Das K, Arnold E, Ebright RH Molecular Cell 66, 169-179, 2017
Affinity selection-mass spectrometry identifies a novel antibacterial RNA polymerase inhibitor
Walker, SS, Degen D, Nickbarg E, Carr D, Soriano A, Mandal M B, Painter RE, Sheth PR, Xiao L, Sher X, Murgolo N, Su J, Olsen DB, Ebright RH, Young K. 2017.
ACS Chemical Biology 2, 1346-1352, 2017
The mechanism of RNA 5′ capping with NAD+, NADH and desphospho-CoA
Bird J, Zhang Y, Tian Y, Panova N, Barvík I, Greene, L Liu M, Buckley B, Krásný L, Lee JK, Kaplan CD, Ebright RH, Nickels BE
Nature 535, 444-447, 2016
Structural basis of transcription activation
Feng Y, Zhang Y, Ebright RH Science 352, 1330-1333, 2016
Multiplexed protein-DNA cross-linking: scrunching in transcription start site selection
Winkelman J, Vvedenskaya I, Zhang Y, Zhang Y, Bird J, Taylor D, Gourse R, Ebright RH, Nickels B Science 351, 1090-1093, 2016.
Structural basis of transcription inhibition by CBR hydroxamidines and CBR pyrazoles
Feng Y, Degen D, Wang X, Gigliotti M, Liu S, Zhang Y, Das D, Michalchuk T, Ebright YW, Talaue M, Connell N, Ebright RH Structure 23, 1470-1481, 2015.
Interactions between RNA polymerase and the ''core recognition element" counteract pausing
Vvedenskaya I, Vahedian-Movahed H, Bird J, Knoblauch J, Goldman S, Zhang Y, Ebright RH, Nickels B Science 344, 1285-1289, 2014
Transcription inhibition by the depsipeptide antibiotic salinamide A
Degen D, Feng Y, Zhang Y, Ebright K, Ebright Y, Gigliotti M, Vahedian-Movahed H, Mandal S, Talaue M, Connell N, Arnold E, Fenical W, Ebright, RH eLife, 3, e02451, 2014
GE23077 binds to the RNA polymerase ‘i’ and ‘i+1’ sites and prevents the binding of initiating nucleotides
Zhang Y, Degen D, Ho M, Sineva E, Ebright K, Ebright Y, Mekler V, Vahedian-Movahed H, Feng Y, Yin R, Tuske, S, Irschik H, Jansen R, Maffioli S, Donadio S, Arnold E, and Ebright RH eLife 3, e02450, 2014
Structural basis of transcription initiation
Zhang Y, Feng Y, Chatterjee S, Tuske S, Ho M, Arnold E, and Ebright RH Science 338, 1076-1080, 2012
Opening and closing of the bacterial RNA polymerase clamp
Chakraborty A, Wang D, Ebright Y, Korlann Y, Kortkhonjia E, Kim T, Chowdhury S, Wigneshweraraj S, Irschik H, Jansen R, Nixon BT, Knight J, Weiss S, Ebright RH Science 337, 591-595, 2012
Transcription initiation in bacteria requires RNA polymerase (RNAP) and the transcription initiation factor sigma. The bacterial transcription initiation complex contains six polypeptides (five in RNAP, one in sigma) and promoter DNA, and has a molecular mass of 0.5 MDa.
Understanding bacterial transcription initiation requires 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.
We are using x-ray crystallography and cryo electrom microscopy (cryo-EM) to determine high-resolution structures of transcription initiation complexes, 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 and photocrosslinkers at specific sites within large multisubunit nucleoprotein complexes, and we are developing automated docking algorithms to integrate structural, biophysical, biochemical, and genetic data in order to construct models for structures of complexes.
Transcription complexes are molecular machines that carry out complex, multistep reactions in transcription initiation and elongation:
- RNA polymerase (RNAP) binds to promoter DNA, to yield an RNAP-promoter closed complex.
- 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.
- 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.
- After RNAP synthesizes an RNA product ~10-15 nucleotides in length, RNAP breaks its interactions with promoter DNA, breaks at least some of its interactions with sigma, 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 sigma 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 subdivided into four sub-steps: (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 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 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 magnetic-tweezers DNA nanomanipulation, single-molecule nanopore-tweezers 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.
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 RNA polymerase (RNAP) alpha-subunit C-terminal domain that facilitates closed-complex formation. Activation at gal involves this same interaction and also interactions between CAP and the RNAP alpha-subunit N-terminal domain, and between CAP and sigma, that facilitate isomerization of closed complex to open complex.
Together with collaborators, we are using electron microscopy, x-ray crystallography, and NMR 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.
Bacterial RNA polymerase (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--rifampin, rifapentine, rifabutin, and rifamixin--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 extremely promising: they exhibit potent activity against a broad spectrum of bacterial pathogens (including Staphylococcus aureus MSSA, Staphylococcus aureus MRSA, Enterococcus faecalis, Enterococcus faecium, Clostridium difficile, Mycobacterium tuberculosis, Bacillus anthracis, Francisella tularensis, Burkholderia mallei, and Burkholderia pseudomallei) 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.
Structural biology: x-ray crystallography and cryo-EM
- Structures of pathogen RNA polymerases
- Mycobacterium tuberculosis RNA polymerase
- Staphylococcus aureus RNA polymerase
- Structures of transcription initiation complexes
- RNA polymerase-promoter closed complexes
- RNA polymerase-promoter open complexes
- RNA polymerase-promoter initial transcribing complexes
- Structures of transcription activation complexes
- Class I transcription activation complexes
- Class II transcription activation complexes
- Class III transcription activation complexes
Single-molecule biophysics: fluorescence spectroscopy, magnetic tweezers, nanopore tweezers
- Mechanism of transcription initiation
- DNA loading
- DNA unwinding
- RNA polymerase clamp opening
- RNA polymerase clamp closing
- Sigma displacement
- DNA scrunching in start-site selection
- DNA scrunching in initial transcription
- Core recognition element (CRE) interactions in initiation
- Mechanism of transcription elongation
- RNA polymerase trigger-loop conformation
- RNA polymerase clamp opening in pausing
- DNA scrunching in pausing
- Core recognition element (CRE) interactions in pausing
- Mechanism of transcription termination
- RNA polymerase clamp opening in termination
- RNA polymerase forward translocation in termination
- RNA slippage in termination
- Core recognition element (CRE) interactions in termination
Drug discovery: in vitro screening, in silico screening, medicinal chemistry
- Identification and characterization of inhibitors of bacterial RNA polymerase
- Lariat peptides
- Nucleoside analogs.
- Engineering of biosynthetic loci for inhibitors of bacterial RNA polymerase
- Myxopyronin biosynthetic locus
- Lipiarmycin biosynthetic locus
- Lariat-peptide biosynthetic loci
- Synthesis of improved inhibitors of bacterial RNA polymerase
- Myxopyronin analogs
- Lipiarmycin analogs
- Lariat-peptide analogs
- Arylpropionyl-phloroglucinol analogs
- Aroyl-aryl-phenylalaninamide analogs
- Pseudouridimycin analogs
- Nucleoside analogs
- Bipartite inhibitors
Dr. Richard Ebright
Richard H. Ebright, Ph.D., is Board of Governors Professor of Chemistry and Chemical Biology at Rutgers University and Laboratory Director at the Waksman Institute of Microbiology. He directs a laboratory of approximately fifteen postdoctoral associates, graduate students, and technicians and serves as project leader on three National Institutes of Health research grants ("Bacterial Transcription Complexes," "Therapeutics for Drug‑Resistant Bacteria: Pseudouridimycins," and "Therapeutics for Drug-Resistant Bacteria: Arylpropionyl Phloroglucinols").
His research focusses on the structure, mechanism, and regulation of bacterial transcription complexes, and on the development of inhibitors of bacterial transcription as antituberculosis agents and broad-spectrum antibacterial agents. His research employs tools of structural biology, biophysics, and drug-discovery.
He received his A.B. (Biology, summa cum laude) and Ph.D. (Microbiology and Molecular Genetics) degrees from Harvard University. He performed graduate research at Harvard and the Institut Pasteur and was a Junior Fellow of the Harvard University Society of Fellows. In 1987, he was appointed as a Laboratory Director at the Waksman Institute and a faculty member at Rutgers University. From 1997 to 2013, he was co-appointed as an Investigator of the Howard Hughes Medical Institute.
He has received the Searle Scholar Award, the Walter J. Johnson Prize, the Schering-Plough Award of the American Society for Biochemistry and Molecular Biology, the Waksman Award of the Theobold Smith Society, the MERIT Award of the National Institutes of Health, and the Chancellor's Award for Excellence in Research of Rutgers University. He is a Member of the American Academy of Arts and Sciences and a Fellow of the American Association for Advancement of Science, the American Academy of Microbiology, and the Infectious Diseases Society of America.
He has more than one hundred forty publications in peer-reviewed journals and more than forty issued and pending patents.
He served for sixteen years as editor of the Journal of Molecular Biology. He has served on the National Institutes of Health Molecular Biology Study Section and on National Institutes of Health special emphasis panels. He is a member of the Institutional Biosafety Committee of Rutgers University and the Antimicrobial Resistance Committee of the Infectious Diseases Society of America, and he has been a member of the Working Group on Pathogen Security of the state of New Jersey and the Controlling Dangerous Pathogens Project of the Center for International Security Studies.
Dr. David Degen
Dr. Yon W. Ebright
Dr. Ying Zhang
Dr. Wei Lin
Dr. Chengyuan Wang
Dr. Zhou (Joel) Yin
Min Sung Cho