Richard H. Ebright--Images

 

Transcriptional pausing: modulation by sequence-specific interaction between RNA polymerase core enzyme and the “core recognition element” (CRE).

The crystal structure of the bacterial transcription initiation complex reveals that RNA polymerase core enzyme makes a sequence-specific interactions with a guanine nucleotide of promoter DNA, unstacking the guanine, flipping the guanine, and inserting the guanine into a deep protein pocket (left, stick representation of guanine in protein pocket; right, space-filling representation of guanine in protein pocket). Biochemical experiments reveal that an analogous interaction between RNA polymerase core enzyme and a guanine occurs during transcription elongation, where it serves to counteract pausing by promoting the forward translocation of RNA polymerase

[See Vvedenskaya, I., Vahedian-Movahed, H., Bird, J., Knoblauch, J., Goldman, S., Zhang ,Y., Ebright, R., and Nickels, B. Interactions between RNA polymerase and the "core recognition element" counteract pausing. (2014) Science, 344, 1285-1289; see also Zhang, Y., Feng, Y., Chatterjee, S., Tuske, S., Ho, M., Arnold, E., and Ebright, R. (2012) Structural basis of transcription initiation.  Science 338, 1076-108].

 

 

Adjacent binding of the antibiotics rifamycin SV and GE23077; linkage of rifamycin SV and GE23077 to yield a novel "bipartite inhibitor."

(Left panel) Crystal structure of bacterial RNA polymerase (RNAP) bound simultaneously to the antibacterial drug rifamycin SV (RifSV; yellow) and the new antibacterial agent GE23077 (GE; blue). Red surfaces, residues at which substitutions confer RifSV-resistance. Green surfaces, residues at which substitutions confer GE-resistance. Black bar, position of a linker that can be introduced to generate a novel "bipartite inhibitor" with very high potency and very low susceptibility to target-based resistance.

(Right panel) Synthesis of a bipartite inhibitor comprising RifSV linked, through a one-atom linker, to GE ("RifaGE-3")

[See 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, R.H. (2014) GE23077 binds to the RNA polymerase ‘i’ and ‘i+1’ sites and prevents the binding of initiating nucleotides. eLife 3, e02450.] 

 

 

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Crystal structure of the bacterial transcription initiation complex. 

(A) Summary of protein-nucleic-acid interactions.  Black residue numbers and lines, interactions by RNA polymerase (RNAP); green residue numbers and lines; interactions by the transcription initiation factor sigma; blue, -10 element of DNA nontemplate strand; light blue, discriminator element of DNA nontemplate strand; pink, rest of DNA nontemplate strand; red, DNA template strand; magenta, ribodinucleotide primer GpA; violet, active-center Mg2+; asterisks, water-mediated interactions; cyan boxes, bases unstacked and inserted into pockets. Residues are numbered as in E coli RNAP and sigma70. 

(B) Overall structure (RNAP beta' non‑conserved domain omitted for clarity). RNAP, gray; sigma, yellow. Other colors as in A. 

(C) Interactions of RNAP and sigma with the transcription-bubble nontemplate strand, the transcription‑bubble template strand, and downstream dsDNA (RNAP beta subunit and beta' non‑conserved domain omitted for clarity). Colors as in B.

[See Zhang, Y., Feng, Y., Chatterjee, S., Tuske, S., Ho, M., Arnold, E., and Ebright, R. (2012) Structural basis of transcription initiation.  Science 338, 1076-1080.]

Determination of RNAP clamp conformation in solution.

(A) Measurement of single-molecule FRET between fluorescent probes incorporated at the tips of the RNAP betaβ’ pincer (clamp) and the RNAP βbeta pincer. Open (red), partly closed (yellow), and closed (green) RNAP clamp conformational states are as observed in crystal structures.

(B) Incorporation of fluorescent probes at the tips of the RNAP βbeta’ pincer (clamp) and the RNAP betaβ pincer, by unnatural amino acid mutagenesis to incorporate 4 azidophenylalanine at sites of interest in betaβ’ and betaβ subunits, followed by Staudinger ligation to incorporate fluorescent probes at 4 azidophenylalanines in βbeta’ and betaβ subunits, followed by in vitro reconstitution of RNAP from labelled βbeta’ and betaβ subunits and unlabelled αalpha and omegaω subunits.

(C) Relationship between single-molecule FRET efficiencies, E, and RNAP clamp conformational states. The red boxes denote the open (red), closed (green), and collapsed (blue) clamp states observed in this work for RNAP in solution.

[See Chakraborty, A., Wang, D., Ebright, Y., Korlann, Y., Kortkhonjia, E., Kim, T., Chowdhury, S., Wigneshweraraj, S., Irschik, H., Jansen, R., Nixon, B.T., Knight, J., Weiss, S., and Ebright, R. (2012) Opening and closing of the bacterial RNA polymerase clamp. Science 337, 591-595.]