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 

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
EMAIL: ebright@waksman.rutgers.edu

 

Dr. Richard H. Ebright