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.
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 YW, 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.
New target for inhibition of bacterial RNA polymerase: 'switch region'
Srivastava A, Talaue M, Liu S, Degen D, Ebright RY, Sineva E, Chakraborty A, Druzhinin SY, Chatterjee S, Mukhopadhyay J, Ebright YW, Zozula A, Shen J, Sengupta S, Niedfeldt RR, Xin C, Kaneko T, Irschik H, Jansen R, Donadio S, Connell N, Ebright RH.
Curr. Opin. Microbiol. 14:532–543, 2011
A new drug target - the 'switch region' - has been identified within bacterial RNA polymerase (RNAP), the enzyme that mediates bacterial RNA synthesis. The new target serves as the binding site for compounds that inhibit bacterial RNA synthesis and kill bacteria. Since the new target is present in most bacterial species, compounds that bind to the new target are active against a broad spectrum of bacterial species. Since the new target is different from targets of other antibacterial agents, compounds that bind to the new target are not cross-resistant with other antibacterial agents. Four antibiotics that function through the new target have been identified: myxopyronin, corallopyronin, ripostatin, and lipiarmycin. This review summarizes the switch region, switch-region inhibitors, and implications for antibacterial drug discovery.
Dr. Richard H. Ebright
190 Frelinghuysen Road
Piscataway, NJ 08854