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 R R, Xin C, Kaneko T, Irschik H, Jansen R, Donadio S, Connell N, Ebright RH. 2011.

Journal:

Current opinion in microbiology

Volume Number:

14

Pages:

532-43

Abstract:

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.

Citation:: Srivastava, A, Talaue M, Liu S, Degen D, Ebright RY, Sineva E, Chakraborty A, Druzhinin SY, Chatterjee S, Mukhopadhyay J et al.. 2011. New target for inhibition of bacterial RNA polymerase: 'switch region'. Current opinion in microbiology. 14:532-43.

Dr. Richard H. Ebright

Principal Investigator

Recent Publications

Robb, NC, Cordes T, Hwang L C, Gryte K, Duchi D, Craggs TD, Santoso Y, Weiss S, Ebright RH, Kapanidis AN. 2013. The transcription bubble of the RNA polymerase-promoter open complex exhibits conformational heterogeneity and millisecond-scale dynamics: implications for transcription start-site selection.. Journal of molecular biology. 425:875-885. Abstract

Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts ~14 base-pairs around the transcription start site, and forms a single-stranded "transcription bubble" within a catalytically active RNAP-DNA open complex (RP(o)). There is significant flexibility in the transcription start site, which causes variable spacing between the promoter elements and the start site; this in turn causes differences in the length and sequence at the 5' end of RNA transcripts, and can be important for gene regulation. The start-site variability also implies the presence of some flexibility in the positioning of the DNA relative to the RNAP active site in RP(o). The flexibility may occur in the positioning of the transcription bubble prior to RNA synthesis and may reflect bubble expansion ("scrunching") or bubble contraction ("unscrunching"). Here, we assess the presence of dynamic flexibility in RP(o) with single-molecule Förster Resonance Energy Transfer. We obtain experimental evidence for dynamic flexibility in RP(o) using different FRET rulers and labelling positions. An analysis of FRET distributions of RP(o) using burst variance analysis reveals conformational fluctuations in RP(o) in the millisecond timescale. Further experiments using subsets of nucleotides and DNA mutations allowed us to reprogram the transcription start sites, in a way that can be described by repositioning of the single-stranded transcription bubble relative to the RNAP active site within RP(o). Our study marks the first experimental observation of conformational dynamics in the transcription bubble of RP(o) and indicates that DNA dynamics within the bubble affect the search for transcription start sites.

Chakraborty, A, Wang D, Ebright YW, Korlann Y, Kortkhonjia E, Kim T, Chowdhury S, Wigneshweraraj S, Irschik H, Jansen R et al.. 2012. Opening and closing of the bacterial RNA polymerase clamp.. Science (New York, N.Y.). 337(6094):591-5. AbstractWebsite

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.

Zhang, Y, Feng Y, Chatterjee S, Tuske S, Ho MX, Arnold E, Ebright RH. 2012. Structural Basis of Transcription Initiation.. Science (New York, N.Y.). 338(6110):1076-1080. AbstractWebsite

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, σ(A), and a promoter DNA fragment corresponding to the transcription bubble and downstream dsDNA of the RNAP-promoter open complex. The structures show that σ 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 σ and template-strand ssDNA preorganize template-strand ssDNA to engage the RNAP active center.

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