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.
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.
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.
Dr. Richard H. Ebright
190 Frelinghuysen Road
Piscataway, NJ 08854