Richard H. Ebright--Transcription: Structure, Mechanism, Regulation, and Antibacterial Drug Discovery

Research Summary

Transcription--the 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.  Richard Ebright's laboratory 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.


Featured Publications

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
Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which kills 1.8 million annually. Mtb RNA polymerase (RNAP) is the target of the first-line antituberculosis drug rifampin (Rif). We report crystal structures of Mtb RNAP, alone and in complex with Rif, at 3.8–4.4 Å resolution. The results identify an Mtb-specific structural module of Mtb RNAP and establish that Rif functions by a steric-occlusion mechanism that prevents extension of RNA. We also report non-Rif-related compounds—Nα-aroyl-N-aryl-phenylalaninamides (AAPs)—that potently and selectively inhibit Mtb RNAP and Mtb growth, and we report crystal structures of Mtb RNAP in complex with AAPs. AAPs bind to a different site on Mtb RNAP than Rif, exhibit no cross-resistance with Rif, function additively when co-administered with Rif, and suppress resistance emergence when co-administered with Rif.
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, 2017 (in press; doi: 10.1021/acschembio.6b01133)
The growing prevalence of drug resistant bacteria is a significant global threat to human health. The antibacterial drug rifampin, which functions by inhibiting bacterial RNA polymerase (RNAP), is an important part of the antibacterial armamentarium. Here, in order to identify novel inhibitors of bacterial RNAP, we used affinity-selection mass spectrometry to screen a chemical library for compounds that bind to Escherichia coli RNAP. We identified a novel small molecule, MRL-436, that binds to RNAP, inhibits RNAP, and exhibits antibacterial activity. MRL-436 binds to RNAP through a binding site that differs from the rifampin binding site, inhibits rifampin-resistant RNAP derivatives, and exhibits antibacterial activity against rifampin-resistant strains. Isolation of mutants resistant to the antibacterial activity of MRL-436 yields a missense mutation in codon 622 of the rpoC gene encoding RNAP β′ subunit or a null mutation in the rpoZ gene encoding RNAP ω subunit, confirming that RNAP is the functional cellular target for the antibacterial activity of MRL-436, and indicating that RNAP β′ subunit residue 622 and RNAP ω subunit are required for the antibacterial activity of MRL-436. Similarity between the resistance determinant for MRL-436 and the resistance determinant for the cellular alarmone ppGpp suggests a possible similarity in binding site and/or induced conformational state for MRL-436 and ppGpp.
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 (
The chemical nature of the 5′ end of RNA is a key determinant of RNA stability, processing, localization and translation efficiency [1,2], and has been proposed to provide a layer of ‘epitranscriptomic’ gene regulation3. Recently it has been shown that some bacterial RNA species carry a 5′-end structure reminiscent of the 5′ 7-methylguanylate ‘cap’ in eukaryotic RNA. In particular, RNA species containing a 5′-end nicotinamide adenine dinucleotide (NAD+) or 3′-desphospho-coenzyme A (dpCoA) have been identified in both Gram-negative and Gram-positive bacteria [3,4,5,6]. It has been proposed that NAD+, reduced NAD+ (NADH) and dpCoA caps are added to RNA after transcription initiation, in a manner analogous to the addition of 7-methylguanylate caps [6,7,8]. Here we show instead that NAD+, NADH and dpCoA are incorporated into RNA during transcription initiation, by serving as non-canonical initiating nucleotides (NCINs) for de novo transcription initiation by cellular RNA polymerase (RNAP). We further show that both bacterial RNAP and eukaryotic RNAP II incorporate NCIN caps, that promoter DNA sequences at and upstream of the transcription start site determine the efficiency of NCIN capping, that NCIN capping occurs in vivo, and that NCIN capping has functional consequences. We report crystal structures of transcription initiation complexes containing NCIN-capped RNA products. Our results define the mechanism and structural basis of NCIN capping, and suggest that NCIN-mediated ‘ab initio capping’ may occur in all organisms.

Structural basis of transcription activation

Feng Y, Zhang Y, Ebright RH
Science, 352, 1330-1333, 2016
Class II transcription activators function by binding to a DNA site overlapping a core promoter and stimulating isomerization of an initial RNA polymerase (RNAP)–promoter closed complex into a catalytically competent RNAP-promoter open complex. Here, we report a 4.4 angstrom crystal structure of an intact bacterial class II transcription activation complex. The structure comprises Thermus thermophilus transcription activator protein TTHB099 (TAP) [homolog of Escherichia coli catabolite activator protein (CAP)], T. thermophilus RNAP σA holoenzyme, a class II TAP-dependent promoter, and a ribotetranucleotide primer. The structure reveals the interactions between RNAP holoenzyme and DNA responsible for transcription initiation and reveals the interactions between TAP and RNAP holoenzyme responsible for transcription activation. The structure indicates that TAP stimulates isomerization through simple, adhesive, stabilizing protein-protein interactions with RNAP holoenzyme.
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.
In bacterial transcription initiation, RNA polymerase (RNAP) selects a transcription start site (TSS) at variable distances downstream of core promoter elements. Using next-generation sequencing and unnatural amino acid-mediated protein-DNA cross-linking, we have determined, for a library of 4(10) promoter sequences, the TSS, the RNAP leading-edge position, and the RNAP trailing-edge position. We find that a promoter element upstream of the TSS, the "discriminator," participates in TSS selection, and that, as the TSS changes, the RNAP leading-edge position changes, but the RNAP trailing-edge position does not change. Changes in the RNAP leading-edge position, but not the RNAP trailing-edge position, are a defining hallmark of the "DNA scrunching" that occurs concurrent with RNA synthesis in initial transcription. We propose that TSS selection involves DNA scrunching prior to RNA synthesis.
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.
CBR hydroxamidines are small-molecule inhibitors of bacterial RNA polymerase (RNAP) discovered through high-throughput screening of synthetic-compound libraries. CBR pyrazoles are structurally related RNAP inhibitors discovered through scaffold hopping from CBR hydroxamidines. CBR hydroxamidines and pyrazoles selectively inhibit Gram-negative bacterial RNAP and exhibit selective antibacterial activity against Gram-negative bacteria. Here, we report crystal structures of the prototype CBR hydroxamidine, CBR703, and a CBR pyrazole in complex with E. coli RNAP holoenzyme. In addition, we define the full resistance determinant for CBR703, show that the binding site and resistance determinant for CBR703 do not overlap the binding sites and resistance determinants of other characterized RNAP inhibitors, show that CBR703 exhibits no or minimal cross-resistance with other characterized RNAP inhibitors, and show that co-administration of CBR703 with other RNAP inhibitors results in additive antibacterial activities. The results set the stage for structure-based optimization of CBR inhibitors as antibacterial drugs.
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 elongation is interrupted by sequences that inhibit nucleotide addition and cause RNA polymerase (RNAP) to pause. Here, by use of native elongating transcript sequencing (NET-seq) and a variant of NET-seq that enables analysis of mutant RNAP derivatives in merodiploid cells (mNET-seq), we analyze transcriptional pausing genome-wide in vivo in Escherichia coli. We identify a consensus pause-inducing sequence element, G-10Y-1G+1 (where -1 corresponds to the position of the RNA 3′ end). We demonstrate that sequence-specific interactions between RNAP core enzyme and a core recognition element (CRE) that stabilize transcription initiation complexes also occur in transcription elongation complexes and facilitate pause read-through by stabilizing RNAP in a posttranslocated register. Our findings identify key sequence determinants of transcriptional pausing and establish that RNAP-CRE interactions modulate pausing.

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
We report that bacterial RNA polymerase (RNAP) is the functional cellular target of the depsipeptide antibiotic salinamide A (Sal), and we report that Sal inhibits RNAP through a novel binding site and mechanism. We show that Sal inhibits RNA synthesis in cells and that mutations that confer Sal-resistance map to RNAP genes. We show that Sal interacts with the RNAP active-center 'bridge-helix cap,' comprising the 'bridge-helix N-terminal hinge,' 'F-loop,' and 'link region.' We show that Sal inhibits nucleotide addition in transcription initiation and elongation. We present a crystal structure that defines interactions between Sal and RNAP and effects of Sal on RNAP conformation. We propose that Sal functions by binding to the RNAP bridge-helix cap and preventing conformational changes of the bridge-helix N-terminal hinge necessary for nucleotide addition. The results provide a target for antibacterial drug discovery and a reagent to probe conformation and function of the bridge-helix N-terminal hinge.

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
Using a combination of genetic, biochemical, and structural approaches, we show that the cyclic-peptide antibiotic GE23077 (GE) binds directly to the bacterial RNA polymerase (RNAP) active-center ‘i’ and ‘i+1’ nucleotide binding sites, preventing the binding of initiating nucleotides, and thereby preventing transcription initiation. The target-based resistance spectrum for GE is unusually small, reflecting the fact that the GE binding site on RNAP includes residues of the RNAP active center that cannot be substituted without loss of RNAP activity. The GE binding site on RNAP is different from the rifamycin binding site. Accordingly, GE and rifamycins do not exhibit cross-resistance, and GE and a rifamycin can bind simultaneously to RNAP. The GE binding site on RNAP is immediately adjacent to the rifamycin binding site. Accordingly, covalent linkage of GE to a rifamycin provides a bipartite inhibitor having very high potency and very low susceptibility to target-based resistance.

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
During transcription initiation, RNA polymerase (RNAP) binds and unwinds promoter DNA to form an RNAP-promoter open complex. We have determined crystal structures at 2.9 and 3.0 Å resolution of functional transcription initiation complexes comprising Thermus thermophilus RNA polymerase, sigma A, and a promoter DNA fragment corresponding to the transcription bubble and downstream dsDNA of the RNAP-‑promoter open complex. The structures show that sigma recognizes the -10 element and discriminator element through interactions that include the unstacking and insertion into pockets of three DNA bases, and that RNAP recognizes the ‑-4/+2 region through interactions that include the unstacking and insertion into a pocket of the +2 base. The structures further show that interactions between sigma and template-‑strand ssDNA pre‑organize template-strand ssDNA to engage the RNAP active center.
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
Using single-molecule fluorescence resonance energy transfer, we have defined bacterial RNA polymerase (RNAP) clamp conformation at each step in transcription initiation and elongation. We find that the clamp predominantly is open in free RNAP and early intermediates in transcription initiation but closes upon formation of a catalytically competent transcription initiation complex and remains closed during initial transcription and transcription elongation. We show that four RNAP inhibitors interfere with clamp opening. We propose that clamp opening allows DNA to be loaded into and unwound in the RNAP active-center cleft, that DNA loading and unwinding trigger clamp closure, and that clamp closure accounts for the high stability of initiation complexes and the high stability and processivity of elongation complexes.


Contact Information

Dr. Richard H. Ebright
Waksman Institute
Rutgers University
190 Frelinghuysen Road
Piscataway, NJ 08854
PH: 848-445-5179
FAX: 732-445-5735


Dr. Richard H. Ebright