Publications

2005
Tuske, S, Sarafianos SG, Wang X, Hudson B, Sineva E, Mukhopadhyay J, Birktoft JJ, Leroy O, Ismail S, Clark AD et al..  2005.  Inhibition of bacterial RNA polymerase by streptolydigin: stabilization of a straight-bridge-helix active-center conformation.. Cell. 122(4):541-52. Abstract
We define the target, mechanism, and structural basis of inhibition of bacterial RNA polymerase (RNAP) by the tetramic acid antibiotic streptolydigin (Stl). Stl binds to a site adjacent to but not overlapping the RNAP active center and stabilizes an RNAP-active-center conformational state with a straight-bridge helix. The results provide direct support for the proposals that alternative straight-bridge-helix and bent-bridge-helix RNAP-active-center conformations exist and that cycling between straight-bridge-helix and bent-bridge-helix RNAP-active-center conformations is required for RNAP function. The results set bounds on models for RNAP function and suggest strategies for design of novel antibacterial agents.
Lee, N K, Kapanidis AN, Wang Y, Michalet X, Mukhopadhyay J, Ebright RH, Weiss S.  2005.  Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation.. Biophysical journal. 88(4):2939-53. Abstract
Fluorescence resonance energy transfer (FRET) between a donor (D) and an acceptor (A) at the single-molecule level currently provides qualitative information about distance, and quantitative information about kinetics of distance changes. Here, we used the sorting ability of confocal microscopy equipped with alternating-laser excitation (ALEX) to measure accurate FRET efficiencies and distances from single molecules, using corrections that account for cross-talk terms that contaminate the FRET-induced signal, and for differences in the detection efficiency and quantum yield of the probes. ALEX yields accurate FRET independent of instrumental factors, such as excitation intensity or detector alignment. Using DNA fragments, we showed that ALEX-based distances agree well with predictions from a cylindrical model of DNA; ALEX-based distances fit better to theory than distances obtained at the ensemble level. Distance measurements within transcription complexes agreed well with ensemble-FRET measurements, and with structural models based on ensemble-FRET and x-ray crystallography. ALEX can benefit structural analysis of biomolecules, especially when such molecules are inaccessible to conventional structural methods due to heterogeneity or transient nature.
2004
Mukhopadhyay, J, Sineva E, Knight J, Levy RM, Ebright RH.  2004.  Antibacterial peptide microcin J25 inhibits transcription by binding within and obstructing the RNA polymerase secondary channel.. Molecular cell. 14(6):739-51. Abstract
The antibacterial peptide microcin J25 (MccJ25) inhibits transcription by bacterial RNA polymerase (RNAP). Biochemical results indicate that inhibition of transcription occurs at the level of NTP uptake or NTP binding by RNAP. Genetic results indicate that inhibition of transcription requires an extensive determinant, comprising more than 50 amino acid residues, within the RNAP secondary channel (also known as the "NTP-uptake channel" or "pore"). Biophysical results indicate that inhibition of transcription involves binding of MccJ25 within the RNAP secondary channel. Molecular modeling indicates that binding of MccJ25 within the RNAP secondary channel obstructs the RNAP secondary channel. We conclude that MccJ25 inhibits transcription by binding within and obstructing the RNAP secondary channel--acting essentially as a "cork in a bottle." Obstruction of the RNAP secondary channel represents an attractive target for drug discovery.
Nickels, BE, Mukhopadhyay J, Garrity SJ, Ebright RH, Hochschild A.  2004.  The sigma 70 subunit of RNA polymerase mediates a promoter-proximal pause at the lac promoter.. Nature structural & molecular biology. 11(6):544-50. Abstract
The sigma(70) subunit of RNA polymerase plays an essential role in transcription initiation. In addition, sigma(70) has a critical regulatory role during transcription elongation at the bacteriophage lambda late promoter, lambda P(R'). At this promoter, sigma(70) mediates a pause in early elongation through contact with a DNA sequence element in the initially transcribed region that resembles a promoter -10 element. Here we provide evidence that sigma(70) also mediates a pause in early elongation at the lac promoter (plac). Like that at lambda P(R'), the pause at plac is facilitated by a sequence element in the initially transcribed region that resembles a promoter -10 element. Using biophysical analysis, we demonstrate that the pause-inducing sequence element at plac stabilizes the interaction between sigma(70) and the remainder of the transcription elongation complex. Bioinformatic analysis suggests that promoter-proximal sigma(70)-dependent pauses may play a role in the regulation of many bacterial promoters.
Renfrow, MB, Naryshkin N, Lewis ML, Chen H-T, Ebright RH, Scott RA.  2004.  Transcription factor B contacts promoter DNA near the transcription start site of the archaeal transcription initiation complex.. The Journal of biological chemistry. 279(4):2825-31. Abstract
Transcription initiation in all three domains of life requires the assembly of large multiprotein complexes at DNA promoters before RNA polymerase (RNAP)-catalyzed transcript synthesis. Core RNAP subunits show homology among the three domains of life, and recent structural information supports this homology. General transcription factors are required for productive transcription initiation complex formation. The archaeal general transcription factors TATA-element-binding protein (TBP), which mediates promoter recognition, and transcription factor B (TFB), which mediates recruitment of RNAP, show extensive homology to eukaryal TBP and TFIIB. Crystallographic information is becoming available for fragments of transcription initiation complexes (e.g. RNAP, TBP-TFB-DNA, TBP-TFIIB-DNA), but understanding the molecular topography of complete initiation complexes still requires biochemical and biophysical characterization of protein-protein and protein-DNA interactions. In published work, systematic site-specific protein-DNA photocrosslinking has been used to define positions of RNAP subunits and general transcription factors in bacterial and eukaryal initiation complexes. In this work, we have used systematic site-specific protein-DNA photocrosslinking to define positions of RNAP subunits and general transcription factors in an archaeal initiation complex. Employing a set of 41 derivatized DNA fragments, each having a phenyl azide photoactivable crosslinking agent incorporated at a single, defined site within positions -40 to +1 of the gdh promoter of the hyperthermophilic marine archaea, Pyrococcus furiosus (Pf), we have determined the locations of PfRNAP subunits PfTBP and PfTFB relative to promoter DNA. The resulting topographical information supports the striking homology with the eukaryal initiation complex and permits one major new conclusion, which is that PfTFB interacts with promoter DNA not only in the TATA-element region but also in the transcription-bubble region, near the transcription start site. Comparison with crystallographic information implicates the PfTFB N-terminal domain in the interaction with the transcription-bubble region. The results are discussed in relation to the known effects of substitutions in the TFB and TFIIB N-terminal domains on transcription initiation and transcription start-site selection.
Lawson, CL, Swigon D, Murakami KS, Darst SA, Berman HM, Ebright RH.  2004.  Catabolite activator protein: DNA binding and transcription activation.. Current opinion in structural biology. 14(1):10-20. Abstract
Recently determined structures of the Escherichia coli catabolite activator protein (CAP) in complex with DNA, and in complex with the RNA polymerase alpha subunit C-terminal domain (alphaCTD) and DNA, have yielded insights into how CAP binds DNA and activates transcription. Comparison of multiple structures of CAP-DNA complexes has revealed the contributions of direct and indirect readout to DNA binding by CAP. The structure of the CAP-alphaCTD-DNA complex has provided the first structural description of interactions between a transcription activator and its functional target within the general transcription machinery. Using the structure of the CAP-alphaCTD-DNA complex, the structure of an RNA polymerase-DNA complex, and restraints from biophysical, biochemical and genetic experiments, it has been possible to construct detailed three-dimensional models of intact class I and class II transcription activation complexes.
Revyakin, A, Ebright RH, Strick TR.  2004.  Promoter unwinding and promoter clearance by RNA polymerase: detection by single-molecule DNA nanomanipulation.. Proceedings of the National Academy of Sciences of the United States of America. 101(14):4776-80. Abstract
By monitoring the end-to-end extension of a mechanically stretched, supercoiled, single DNA molecule, we have been able directly to observe the change in extension associated with unwinding of approximately one turn of promoter DNA by RNA polymerase (RNAP). By performing parallel experiments with negatively and positively supercoiled DNA, we have been able to deconvolute the change in extension caused by RNAP-dependent DNA unwinding (with approximately 1-bp resolution) and the change in extension caused by RNAP-dependent DNA compaction (with approximately 5-nm resolution). We have used this approach to quantify the extent of unwinding and compaction, the kinetics of unwinding and compaction, and effects of supercoiling, sequence, ppGpp, and nucleotides. We also have used this approach to detect promoter clearance and promoter recycling by successive RNAP molecules. We find that the rate of formation and the stability of the unwound complex depend profoundly on supercoiling and that supercoiling exerts its effects mechanically (through torque), and not structurally (through the number and position of supercoils). The approach should permit analysis of other nucleic-acid-processing factors that cause changes in DNA twist and/or DNA compaction.
2003
Bayro, MJ, Mukhopadhyay J, Swapna GVT, Huang JY, Ma L-C, Sineva E, Dawson PE, Montelione GT, Ebright RH.  2003.  Structure of antibacterial peptide microcin J25: a 21-residue lariat protoknot.. Journal of the American Chemical Society. 125(41):12382-3. Abstract
The antibacterial peptide microcin J25 (MccJ25) inhibits bacterial transcription by binding within, and obstructing, the nucleotide-uptake channel of bacterial RNA polymerase. Published covalent and three-dimensional structures indicate that MccJ25 is a 21-residue cycle. Here, we show that the published covalent and three-dimensional structures are incorrect, and that MccJ25 in fact is a 21-residue "lariat protoknot", consisting of an 8-residue cyclic segment followed by a 13-residue linear segment that loops back and threads through the cyclic segment. MccJ25 is the first example of a lariat protoknot involving a backbone-side chain amide linkage.
Chen, H, Tang H, Ebright RH.  2003.  Functional interaction between RNA polymerase alpha subunit C-terminal domain and sigma70 in UP-element- and activator-dependent transcription.. Molecular cell. 11(6):1621-33. Abstract
We show that the Escherichia coli RNA polymerase (RNAP) alpha subunit C-terminal domain (alphaCTD) functionally interacts with sigma(70) at a subset of UP-element- and activator-dependent promoters, we define the determinants of alphaCTD and sigma(70) required for the interaction, and we present a structural model for the interaction. The alphaCTD-sigma(70) interaction spans the upstream promoter and core promoter, thereby linking recognition of UP-elements and activators in the upstream promoter with recognition of the -35 element in the core promoter. We propose that the alphaCTD-sigma(70) interaction permits UP-elements and activators not only to "recruit" RNAP through direct interaction with alphaCTD, but also to "remodel" RNAP-core-promoter interaction through indirect, alphaCTD-bridged interactions with sigma(70).
Mukhopadhyay, J, Mekler V, Kortkhonjia E, Kapanidis AN, Ebright YW, Ebright RH.  2003.  Fluorescence resonance energy transfer (FRET) in analysis of transcription-complex structure and function.. Methods in enzymology. 371:144-59.
Revyakin, A, Allemand JF, Croquette V, Ebright RH, Strick TR.  2003.  Single-molecule DNA nanomanipulation: detection of promoter-unwinding events by RNA polymerase.. Methods in enzymology. 370:577-98.
2002
Lloyd, GS, Niu W, Tebbutt J, Ebright RH, Busby SJW.  2002.  Requirement for two copies of RNA polymerase alpha subunit C-terminal domain for synergistic transcription activation at complex bacterial promoters.. Genes & development. 16(19):2557-65. Abstract
Transcription activation by the Escherichia coli cyclic AMP receptor protein (CRP) at different promoters has been studied using RNA polymerase holoenzyme derivatives containing two full-length alpha subunits, or containing one full-length alpha subunit and one truncated alpha subunit lacking the alpha C-terminal domain (alpha CTD). At a promoter having a single DNA site for CRP, activation requires only one full-length alpha subunit. Likewise, at a promoter having a single DNA site for CRP and one adjacent UP-element subsite (high-affinity DNA site for alpha CTD), activation requires only one full-length alpha subunit. In contrast, at promoters having two DNA sites for CRP, or one DNA site for CRP and two UP-element subsites, activation requires two full-length alpha subunits. We conclude that a single copy of alpha CTD is sufficient to interact with one CRP molecule and one adjacent UP-element subsite, but two copies of alpha CTD are required to interact with two CRP molecules or with one CRP molecule and two UP-element subsites.
Mekler, V, Kortkhonjia E, Mukhopadhyay J, Knight J, Revyakin A, Kapanidis AN, Niu W, Ebright YW, Levy R, Ebright RH.  2002.  Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex.. Cell. 108(5):599-614. Abstract
We have used systematic fluorescence resonance energy transfer and distance-constrained docking to define the three-dimensional structures of bacterial RNA polymerase holoenzyme and the bacterial RNA polymerase-promoter open complex in solution. The structures provide a framework for understanding sigma(70)-(RNA polymerase core), sigma(70)-DNA, and sigma(70)-RNA interactions. The positions of sigma(70) regions 1.2, 2, 3, and 4 are similar in holoenzyme and open complex. In contrast, the position of sigma(70) region 1.1 differs dramatically in holoenzyme and open complex. In holoenzyme, region 1.1 is located within the active-center cleft, apparently serving as a "molecular mimic" of DNA, but, in open complex, region 1.1 is located outside the active center cleft. The approach described here should be applicable to the analysis of other nanometer-scale complexes.
Ebright, RH, Connell ND.  2002.  Bioweapon agents: more access means more risk.. Nature. 415(6870):364.
Benoff, B, Yang H, Lawson CL, Parkinson G, Liu J, Blatter E, Ebright YW, Berman HM, Ebright RH.  2002.  Structural basis of transcription activation: the CAP-alpha CTD-DNA complex.. Science (New York, N.Y.). 297(5586):1562-6. Abstract
The Escherichia coli catabolite activator protein (CAP) activates transcription at P(lac), P(gal), and other promoters through interactions with the RNA polymerase alpha subunit carboxyl-terminal domain (alphaCTD). We determined the crystal structure of the CAP-alphaCTD-DNA complex at a resolution of 3.1 angstroms. CAP makes direct protein-protein interactions with alphaCTD, and alphaCTD makes direct protein-DNA interactions with the DNA segment adjacent to the DNA site for CAP. There are no large-scale conformational changes in CAP and alphaCTD, and the interface between CAP and alphaCTD is small. These findings are consistent with the proposal that activation involves a simple "recruitment" mechanism.
Savery, NJ, Lloyd GS, Busby SJW, Thomas MS, Ebright RH, Gourse RL.  2002.  Determinants of the C-terminal domain of the Escherichia coli RNA polymerase alpha subunit important for transcription at class I cyclic AMP receptor protein-dependent promoters.. Journal of bacteriology. 184(8):2273-80. Abstract
Alanine scanning of the Escherichia coli RNA polymerase alpha subunit C-terminal domain (alphaCTD) was used to identify amino acid side chains important for class I cyclic AMP receptor protein (CRP)-dependent transcription. Key residues were investigated further in vivo and in vitro. Substitutions in three regions of alphaCTD affected class I CRP-dependent transcription from the CC(-61.5) promoter and/or the lacP1 promoter. These regions are (i) the 287 determinant, previously shown to contact CRP during class II CRP-dependent transcription; (ii) the 265 determinant, previously shown to be important for alphaCTD-DNA interactions, including those required for class II CRP-dependent transcription; and (iii) the 261 determinant. We conclude that CRP contacts the same target in alphaCTD, the 287 determinant, at class I and class II CRP-dependent promoters. We also conclude that the relative contributions of individual residues within the 265 determinant depend on promoter sequence, and we discuss explanations for effects of substitutions in the 261 determinant.
2001
Kapanidis, AN, Ebright YW, Ludescher RD, Chan S, Ebright RH.  2001.  Mean DNA bend angle and distribution of DNA bend angles in the CAP-DNA complex in solution.. Journal of molecular biology. 312(3):453-68. Abstract
In order to define the mean DNA bend angle and distribution of DNA bend angles in the catabolite activator protein (CAP)-DNA complex in solution under standard transcription initiation conditions, we have performed nanosecond time-resolved fluorescence measurements quantifying energy transfer between a probe incorporated at a specific site in CAP, and a complementary probe incorporated at each of five specific sites in DNA. The results indicate that the mean DNA bend angle is 77(+/-3) degrees - consistent with the mean DNA bend angle observed in crystallographic structures (80(+/-12) degrees ). Lifetime-distribution analysis indicates that the distribution of DNA bend angles is relatively narrow, with <10 % of DNA bend angles exceeding 100 degrees. Millisecond time-resolved luminescence measurements using lanthanide-chelate probes provide independent evidence that the upper limit of the distribution of DNA bend angles is approximately 100 degrees. The methods used here will permit mutational analysis of CAP-induced DNA bending and the role of CAP-induced DNA bending in transcriptional activation.
Chen, S, Gunasekera A, Zhang X, Kunkel TA, Ebright RH, Berman HM.  2001.  Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: alteration of DNA binding specificity through alteration of DNA kinking.. Journal of molecular biology. 314(1):75-82. Abstract
The catabolite activator protein (CAP) sharply bends DNA in the CAP-DNA complex, introducing a DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site, 5'-A(1)A(2)A(3)T(4)G(5)T(6)G(7)A(8)T(9)C(10)T(11)-3' ("primary kink"). CAP recognizes the base-pair immediately 5' to the primary-kink site, T:A(6), through an "indirect-readout" mechanism involving sequence effects on the energetics of primary-kink formation. CAP recognizes the base-pair immediately 3' to the primary-kink site, G:C(7), through a "direct-readout" mechanism involving formation of a hydrogen bond between Glu181 of CAP and G:C(7). Here, we report that substitution of the carboxylate side-chain of Glu181 of CAP by the one-methylene-group-shorter carboxylate side-chain of Asp changes DNA binding specificity at position 6 of the DNA half site, changing specificity for T:A(6) to specificity for C:G(6), and we report a crystallographic analysis defining the structural basis of the change in specificity. The Glu181-->Asp substitution eliminates the primary kink and thus eliminates indirect-readout-based specificity for T:A(6). The Glu181-->Asp substitution does not eliminate hydrogen-bond formation with G:C(7), and thus does not eliminate direct-readout-based specificity for G:C(7).
Chen, S, Vojtechovsky J, Parkinson GN, Ebright RH, Berman HM.  2001.  Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: DNA binding specificity based on energetics of DNA kinking.. Journal of molecular biology. 314(1):63-74. Abstract
The catabolite activator protein (CAP) makes no direct contact with the consensus base-pair T:A at position 6 of the DNA half-site 5'-A(1)A(2)A(3)T(4)G(5)T(6)G(7)A(8)T(9)C(10)T(11)-3' but, nevertheless, exhibits strong specificity for T:A at position 6. Binding of CAP results in formation of a sharp DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site. The consensus base-pair T:A at position 6 and the consensus base-pair G:C at position 7 form a T:A/G:C step, which is known to be associated with DNA flexibility. It has been proposed that specificity for T:A at position 6 is a consequence of formation of the DNA kink between positions 6 and 7, and of effects of the T:A(6)/G:C(7) step on the geometry of DNA kinking, or the energetics of DNA kinking. In this work, we determine crystallographic structures of CAP-DNA complexes having the consensus base-pair T:A at position 6 or the non-consensus base-pair C:G at position 6. We show that complexes containing T:A or C:G at position 6 exhibit similar overall DNA bend angles and local geometries of DNA kinking. We infer that indirect readout in this system does not involve differences in the geometry of DNA kinking but, rather, solely differences in the energetics of DNA kinking. We further infer that the main determinant of DNA conformation in this system is protein-DNA interaction, and not DNA sequence.
Minakhin, L, Bhagat S, Brunning A, Campbell EA, Darst SA, Ebright RH, Severinov K.  2001.  Bacterial RNA polymerase subunit omega and eukaryotic RNA polymerase subunit RPB6 are sequence, structural, and functional homologs and promote RNA polymerase assembly.. Proceedings of the National Academy of Sciences of the United States of America. 98(3):892-7. Abstract
Bacterial DNA-dependent RNA polymerase (RNAP) has subunit composition beta'betaalpha(I)alpha(II)omega. The role of omega has been unclear. We show that omega is homologous in sequence and structure to RPB6, an essential subunit shared in eukaryotic RNAP I, II, and III. In Escherichia coli, overproduction of omega suppresses the assembly defect caused by substitution of residue 1362 of the largest subunit of RNAP, beta'. In yeast, overproduction of RPB6 suppresses the assembly defect caused by the equivalent substitution in the largest subunit of RNAP II, RPB1. High-resolution structural analysis of the omega-beta' interface in bacterial RNAP, and comparison with the RPB6-RPB1 interface in yeast RNAP II, confirms the structural relationship and suggests a "latching" mechanism for the role of omega and RPB6 in promoting RNAP assembly.
Mukhopadhyay, J, Kapanidis AN, Mekler V, Kortkhonjia E, Ebright YW, Ebright RH.  2001.  Translocation of sigma(70) with RNA polymerase during transcription: fluorescence resonance energy transfer assay for movement relative to DNA.. Cell. 106(4):453-63. Abstract
Using fluorescence resonance energy transfer, we show that, in the majority of transcription complexes, sigma(70) is not released from RNA polymerase upon transition from initiation to elongation, but, instead, remains associated with RNA polymerase and translocates with RNA polymerase. The results argue against the presumption that there are necessary subunit-composition differences, and corresponding necessary mechanistic differences, in initiation and elongation. The methods of this report should be generalizable to monitor movement of any molecule relative to any nucleic acid.
Naryshkin, N, Kim Y, Dong Q, Ebright RH.  2001.  Site-specific protein-DNA photocrosslinking. Analysis of bacterial transcription initiation complexes.. Methods in molecular biology (Clifton, N.J.). 148:337-61.
2000
Kim, TK, Ebright RH, Reinberg D.  2000.  Mechanism of ATP-dependent promoter melting by transcription factor IIH.. Science (New York, N.Y.). 288(5470):1418-22. Abstract
We show that transcription factor IIH ERCC3 subunit, the DNA helicase responsible for adenosine triphosphate (ATP)-dependent promoter melting during transcription initiation, does not interact with the promoter region that undergoes melting but instead interacts with DNA downstream of this region. We show further that promoter melting does not change protein-DNA interactions upstream of the region that undergoes melting but does change interactions within and downstream of this region. Our results rule out the proposal that IIH functions in promoter melting through a conventional DNA-helicase mechanism. We propose that IIH functions as a molecular wrench: rotating downstream DNA relative to fixed upstream protein-DNA interactions, thereby generating torque on, and melting, the intervening DNA.
Naryshkin, N, Revyakin A, Kim Y, Mekler V, Ebright RH.  2000.  Structural organization of the RNA polymerase-promoter open complex.. Cell. 101(6):601-11. Abstract
We have used systematic site-specific protein-DNA photocrosslinking to define interactions between bacterial RNA polymerase (RNAP) and promoter DNA in the catalytically competent RNAP-promoter open complex (RPo). We have mapped more than 100 distinct crosslinks between individual segments of RNAP subunits and individual phosphates of promoter DNA. The results provide a comprehensive description of protein-DNA interactions in RPo, permit construction of a detailed model for the structure of RPo, and permit analysis of effects of a transcriptional activator on the structure of RPo.