Richard H. Ebright--Images
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.]
Structural basis of transcription inhibition by myxopyronin: contacts between RNA polymerase and myxopyronin.
(A) Binding pocket for myxopyronin. Cyan, surface representation of the binding pocket and adjacent hydrophobic pocket. Gray, ribbon representation of RNA polymerase backbone. Green, myxopyronin carbon atoms; red, myxopyronin oxygen atoms. RNA polymerase residues are numbered both as in T. thermophilus RNA polymerase and as in E. coli RNA polymerase (in parentheses).
(B) Contacts between RNA polymerase and myxopyronin (stereoview). Gray, RNA polymerase backbone (ribbon representation) and RNA polymerase sidechain carbon atoms (stick representation); green, myxopyronin carbon atoms; red, oxygen atoms; blue, nitrogen atoms. "W," ordered bound water molecule. Dashed lines, H-bonds.
(C) Schematic summary of contacts between RNA polymerase and myxopyronin. "W", ordered bound water molecule. Red dashed lines, H-bonds. Blue arcs, van der Waals interactions.
[See Mukhopadhyay, J., Das, K., Ismail, S., Koppstein, D., Jang, M., Hudson, B., Sarafianos, S., Tuske, S., Patel, J., Jansen, R., Irschik, H., Arnold, E., and Ebright, R. (2008) The RNA polymerase "switch region" is a target of inhibitors Cell 135, 295-307.]
RNA polymerase clamp and RNA polymerase switch region.
(A) Conformational states of the RNA polymerase clamp (two orthogonal views). Structure of RNA polymerase showing open (red), partly closed (yellow), and fully closed (green) clamp conformations, as observed in crystal structures (PDB 1I3Q, PDB 1HQM, PDB 1I6H). Circle, switch region; dashed circle, binding site for rifamycins; violet sphere, active-center Mg2+.
(B) Conformational states of the RNA polymerase switch region (stereoview). Structures of switch 1 and switch 2 (beta' residues 1304-1329 and beta' residues 330-349; residues numbered as in E. coli RNA polymerase) showing conformational states associated with open (red), partly closed (yellow), and fully closed (green) clamp conformations, as observed in crystal structures (PDB 1I3Q, PDB 1HQM, PDB 1I6H). Gray squares, points of connection of switch 1 and switch 2 to the RNA polymerase main mass. Colored circles, points of connection of switch 1 and switch 2 to the RNA polymerase clamp.
[See Mukhopadhyay, J., Das, K., Ismail, S., Koppstein, D., Jang, M., Hudson, B., Sarafianos, S., Tuske, S., Patel, J., Jansen, R., Irschik, H., Arnold, E., and Ebright, R. (2008) The RNA polymerase "switch region" is a target of inhibitors Cell 135, 295-307.]
Initial transcription by RNA polymerase proceeds through a "DNA scrunching" mechanism, in which the enzyme remains stationary on promoter DNA and pulls into itself downstream DNA. Proposed movements of the template and nontemplate DNA strands are indicated by blue-outlined and red-outlined arrows. Proposed positions at which the scrunched template and nontemplate DNA strands emerge from the enzyme are indicated by orange and pink dashed lines. Positions of fluorescent probes used to analyze scrunching are indicated in green (donor probe on polymerase), brick red (acceptor probe at promoter position +20 in absence of scrunching), and bright red (acceptor probe at promoter position +20 in presence of scrunching).
[See Revyakin, A., Liu, C., Ebright, R.H. & Strick, T. (2006) Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching. Science 314, 1139-1143; Kapanidis, A., Margeat, E., Ho, S.O., Kortkhonjia, E., Weiss, S. & Ebright, R.H. (2006) Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science 314, 1144-1147.]
Microcin J25 (MccJ25) inhibits bacterial transcription by binding within, and obstructing, the bacterial RNA polymerase nucleotide-uptake channel, acting essentially as a "cork in a bottle." Sites of single-residue substitutions in RNA polymerase that confer MccJ25-resistance are shown in red (beta' subunit) and pink (beta subunit). The RNA polymerase active-center Mg2+ is shown in white.
[See Mukhopadhyay, J., Sineva, E., Knight, J., Levy, R., and Ebright, R. (2004) Antibacterial peptide microcin J25 (MccJ25) inhibits transcription by binding within and obstructing the RNA polymerase secondary channel. Molecular Cell 14, 739–751.]
Model for the structural organization of the bacterial RNA polymerase-promoter open complex, derived from systematic fluorescence resonance energy transfer and distance-constrained docking. Sigma70 regions 2 and 4 are shown as yellow ribbons, with the alpha-helices that mediate recognition of the promoter -10 element and -35 element highlighted in light yellow; sigma70 regions 1.1, 3.1, and 3.2 are shown as yellow spheres. RNAP subunits beta', beta, alphaI, alphaII, and omega are shown in orange, green, light blue, dark blue, and gray. Openings of channels for the nontemplate DNA strand (NT), the template DNA strand (T), and the RNA product (RNA) are indicated in magenta.
[See Mekler, V., Kortkhonjia, E., Mukhopadhyay, J., Knight, J., Revyakin, A., Kapanidis, A., Niu, W., Ebright, Y., Levy, R., and Ebright, R. (2002) Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex. Cell 108, 599-614].
Use of systematic fluorescence resonance energy transfer and distance-constrained docking to define the structural organization of the bacterial RNA polymerase-promoter open complex (two orthogonal views). Measured distances between probes in sigma70 (yellow spheres) and probes in RNA polymerase core and DNA (white spheres) define the positions of segments of sigma70 relative to RNA polymerase core and DNA in the RNA polymerase-promoter open complex in solution. Sigma70 region 2 (sigmaR2), which is responsible for recognition of the promoter -10 element, and for which a crystal structure is available, is shown as a yellow ribbon with probe sites as yellow spheres. Sigma70 region 4 (sigmaR4), which is responsible for recognition of the promoter -35 element, and for which a homology-modelled structure is available, also is shown as a yellow ribbon with probe sites as yellow spheres. Sigma70 regions 1.1, 3.1, and 3.2 (sigmaR1.1, sigmaR3.1, and sigmaR3.2), for which no structural information is available, are shown as yellow spheres. Distances to sigmaR2, to sigmaR4, and to sigmaR1.1, sigmaR3.1, and sigmaR3.2, are shown in, respectively, green, blue, and white. Docking of segments of sigma70 onto RNA polymerase core and DNA was performed using 105 measured distances and an automated distance-constrained-docking algorithm employing only geometric information--i.e., the 105 measured distances, and the relative positions of probe sites. [For clarity, each distance is shown as a single Ca-Ca or Ca -P vector. The actual distance-constrained-docking algorithm used ensembles of probe-probe vectors, reflecting ensembles of probe and linker conformations.]
[See Mekler, V., Kortkhonjia, E., Mukhopadhyay, J., Knight, J., Revyakin, A., Kapanidis, A., Niu, W., Ebright, Y., Levy, R., and Ebright, R. (2002) Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex. Cell 108, 599-614].
Fluorescence resonance energy transfer (FRET) establishes that, in the majority of transcription complexes, sigma70 is not released from RNA polymerase (RNAP) upon transition to elongation, but, instead, is retained by RNAP and translocates with RNAP. The four conserved regions of sigma70--regions 1, 2, 3, and 4--occupy the same, or nearly the same, positions relative to RNAP in the RNAP-promoter open complex (RPo) and in an RNAP-DNA elongation complex containing 11 nt of RNA (RDe,11). Sigma70 regions 1, 2, 3, and 4 are in yellow; RNAP beta', beta, alpha^I, and omega subunits are in orange, green, blue, and gray; DNA template and nontemplate strands are in pink and gray.
[See Mukhopadhyay, J., Kapanidis, A., Mekler, V., Kortkhonjia, E., Ebright, Y., and Ebright, R. Translocation of sigma70 with RNA polymerase during transcription: fluorescence resonance energy transfer assay for movement relative to DNA. Cell 2001 106:453-463.]
Crystal structure of a transcriptional activator (catabolite activator protein, CAP; cyan) in complex with its target in the transcriptional machinery (RNA polymerase alpha-subunit C-terminal domain, alphaCTD; green) and DNA (red). There are no large-scale conformational changes in the activator and target, and the interface between the activator and target is small (residues highlighted in navy and yellow)--consistent with the proposal that transcriptional activation involves a simple "recruitment" mechanism.
[See Benoff, B., Yang, H., Lawson, C., Parkinson, G., Liu, J., Blatter, E., Ebright, Y., Berman, H., and Ebright, R. (2002) Structural basis of transcription activation: the CAP-alphaCTD-DNA complex, Science, 297, 1562-1566.]
