Grohmann, D, Nagy J, Chakraborty A, Klose D, Fielden D, Ebright RH, Michaelis J, Werner F.  2011.  The initiation factor tfe and the elongation factor Spt4/5 compete for the RNAP clamp during transcription initiation and elongation.. Molecular cell. 43(2):263-74. Abstract
TFIIE and the archaeal homolog TFE enhance DNA strand separation of eukaryotic RNAPII and the archaeal RNAP during transcription initiation by an unknown mechanism. We have developed a fluorescently labeled recombinant M. jannaschii RNAP system to probe the archaeal transcription initiation complex, consisting of promoter DNA, TBP, TFB, TFE, and RNAP. We have localized the position of the TFE winged helix (WH) and Zinc ribbon (ZR) domains on the RNAP using single-molecule FRET. The interaction sites of the TFE WH domain and the transcription elongation factor Spt4/5 overlap, and both factors compete for RNAP binding. Binding of Spt4/5 to RNAP represses promoter-directed transcription in the absence of TFE, which alleviates this effect by displacing Spt4/5 from RNAP. During elongation, Spt4/5 can displace TFE from the RNAP elongation complex and stimulate processivity. Our results identify the RNAP "clamp" region as a regulatory hot spot for both transcription initiation and transcription elongation.
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. 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.
Chakraborty, A, Wang D, Ebright YW, Ebright RH.  2010.  Azide-specific labeling of biomolecules by Staudinger-Bertozzi ligation phosphine derivatives of fluorescent probes suitable for single-molecule fluorescence spectroscopy.. Methods in enzymology. 472:19-30. Abstract
We describe the synthesis of phosphine derivatives of three fluorescent probes that have a brightness and photostability suitable for single-molecule fluorescence spectroscopy and microscopy: Alexa488, Cy3B, and Alexa647. In addition, we describe procedures for use of these reagents in azide-specific, bioorthogonal labeling through Staudinger-Bertozzi ligation, as well as procedures for the quantitation of labeling specificity and labeling efficiency. The reagents and procedures of this report enable chemoselective, site-selective labeling of azide-containing biomolecules for single-molecule fluorescence spectroscopy and microscopy.
Hudson, BP, Quispe J, Lara-González S, Kim Y, Berman HM, Arnold E, Ebright RH, Lawson CL.  2009.  Three-dimensional EM structure of an intact activator-dependent transcription initiation complex.. Proceedings of the National Academy of Sciences of the United States of America. 106(47):19830-5. Abstract
We present the experimentally determined 3D structure of an intact activator-dependent transcription initiation complex comprising the Escherichia coli catabolite activator protein (CAP), RNA polymerase holoenzyme (RNAP), and a DNA fragment containing positions -78 to +20 of a Class I CAP-dependent promoter with a CAP site at position -61.5 and a premelted transcription bubble. A 20-A electron microscopy reconstruction was obtained by iterative projection-based matching of single particles visualized in carbon-sandwich negative stain and was fitted using atomic coordinate sets for CAP, RNAP, and DNA. The structure defines the organization of a Class I CAP-RNAP-promoter complex and supports previously proposed interactions of CAP with RNAP alpha subunit C-terminal domain (alphaCTD), interactions of alphaCTD with sigma(70) region 4, interactions of CAP and RNAP with promoter DNA, and phased-DNA-bend-dependent partial wrapping of DNA around the complex. The structure also reveals the positions and shapes of species-specific domains within the RNAP beta', beta, and sigma(70) subunits.
Goldman, SR, Ebright RH, Nickels BE.  2009.  Direct detection of abortive RNA transcripts in vivo.. Science (New York, N.Y.). 324(5929):927-8. Abstract
During transcription initiation in vitro, prokaryotic and eukaryotic RNA polymerase (RNAP) can engage in abortive initiation-the synthesis and release of short (2 to 15 nucleotides) RNA transcripts-before productive initiation. It has not been known whether abortive initiation occurs in vivo. Using hybridization with locked nucleic acid probes, we directly detected abortive transcripts in bacteria. In addition, we show that in vivo abortive initiation shows characteristics of in vitro abortive initiation: Abortive initiation increases upon stabilizing interactions between RNAP and either promoter DNA or sigma factor, and also upon deleting elongation factor GreA. Abortive transcripts may have functional roles in regulating gene expression in vivo.
Ho, MX, Hudson BP, Das K, Arnold E, Ebright RH.  2009.  Structures of RNA polymerase-antibiotic complexes.. Current opinion in structural biology. 19(6):715-23. Abstract
Inhibition of bacterial RNA polymerase (RNAP) is an established strategy for antituberculosis therapy and broad-spectrum antibacterial therapy. Crystal structures of RNAP-inhibitor complexes are available for four classes of antibiotics: rifamycins, sorangicin, streptolydigin, and myxopyronin. The structures define three different targets, and three different mechanisms, for inhibition of bacterial RNAP: (1) rifamycins and sorangicin bind near the RNAP active center and block extension of RNA products; (2) streptolydigin interacts with a target that overlaps the RNAP active center and inhibits conformational cycling of the RNAP active center; and (3) myxopyronin interacts with a target remote from the RNAP active center and functions by interfering with opening of the RNAP active-center cleft to permit entry and unwinding of DNA and/or by interfering with interactions between RNAP and the DNA template strand. The structures enable construction of homology models of pathogen RNAP-antibiotic complexes, enable in silico screening for new antibacterial agents, and enable rational design of improved antibacterial agents.
Popovych, N, Tzeng S-R, Tonelli M, Ebright RH, Kalodimos CG.  2009.  Structural basis for cAMP-mediated allosteric control of the catabolite activator protein.. Proceedings of the National Academy of Sciences of the United States of America. 106(17):6927-32. Abstract
The cAMP-mediated allosteric transition in the catabolite activator protein (CAP; also known as the cAMP receptor protein, CRP) is a textbook example of modulation of DNA-binding activity by small-molecule binding. Here we report the structure of CAP in the absence of cAMP, which, together with structures of CAP in the presence of cAMP, defines atomic details of the cAMP-mediated allosteric transition. The structural changes, and their relationship to cAMP binding and DNA binding, are remarkably clear and simple. Binding of cAMP results in a coil-to-helix transition that extends the coiled-coil dimerization interface of CAP by 3 turns of helix and concomitantly causes rotation, by approximately 60 degrees , and translation, by approximately 7 A, of the DNA-binding domains (DBDs) of CAP, positioning the recognition helices in the DBDs in the correct orientation to interact with DNA. The allosteric transition is stabilized further by expulsion of an aromatic residue from the cAMP-binding pocket upon cAMP binding. The results define the structural mechanisms that underlie allosteric control of this prototypic transcriptional regulatory factor and provide an illustrative example of how effector-mediated structural changes can control the activity of regulatory proteins.
Naryshkin, N, Druzhinin S, Revyakin A, Kim Y, Mekler V, Ebright RH.  2009.  Static and kinetic site-specific protein-DNA photocrosslinking: analysis of bacterial transcription initiation complexes.. Methods in molecular biology (Clifton, N.J.). 543:403-37. Abstract
Static site-specific protein-DNA photocrosslinking permits identification of protein-DNA interactions within multiprotein-DNA complexes. Kinetic site-specific protein-DNA photocrosslinking - involving rapid-quench-flow mixing and pulsed-laser irradiation - permits elucidation of pathways and kinetics of formation of protein-DNA interactions within multiprotein-DNA complexes. We present detailed protocols for application of static and kinetic site-specific protein-DNA photocrosslinking to bacterial transcription initiation complexes.
Feklistov, A, Mekler V, Jiang Q, Westblade LF, Irschik H, Jansen R, Mustaev A, Darst SA, Ebright RH.  2008.  Rifamycins do not function by allosteric modulation of binding of Mg2+ to the RNA polymerase active center.. Proceedings of the National Academy of Sciences of the United States of America. 105(39):14820-5. Abstract
Rifamycin antibacterial agents inhibit bacterial RNA polymerase (RNAP) by binding to a site adjacent to the RNAP active center and preventing synthesis of RNA products >2-3 nt in length. Recently, Artsimovitch et al. [(2005) Cell 122:351-363] proposed that rifamycins function by allosteric modulation of binding of Mg(2+) to the RNAP active center and presented three lines of biochemical evidence consistent with this proposal. Here, we show that rifamycins do not affect the affinity of binding of Mg(2+) to the RNAP active center, and we reassess the three lines of biochemical evidence, obtaining results not supportive of the proposal. We conclude that rifamycins do not function by allosteric modulation of binding of Mg(2+) to the RNAP active center.
Pavlova, O, Mukhopadhyay J, Sineva E, Ebright RH, Severinov K.  2008.  Systematic structure-activity analysis of microcin J25.. The Journal of biological chemistry. 283(37):25589-95. Abstract
Microcin J25 (MccJ25) is a 21-residue plasmid-encoded ribosomally synthesized lariat-protoknot antibacterial peptide that targets bacterial RNA polymerase. MccJ25 consists of an 8-residue cycle followed by a 13-residue tail that loops back and threads through the cycle. We have performed systematic mutational scanning of MccJ25, constructing and analyzing more than 380 singly substituted derivatives of MccJ25. The results define residues important for production of MccJ25 (comprising synthesis of MccJ25 precursor, processing of MccJ25 precursor, export of mature MccJ25, and stability of mature MccJ25), inhibition of RNA polymerase, and inhibition of bacterial growth. The results show that only a small number of residues (three in the cycle and one in the threaded segment of the tail) are important for MccJ25 production. The results further show that only a small number of additional residues (two in the cycle and four in the threaded segment of the tail) are important for inhibition of transcription. The results open the way for design and construction of more potent MccJ25-based inhibitors of bacterial growth.
Mukhopadhyay, J, Das K, Ismail S, Koppstein D, Jang M, Hudson B, Sarafianos S, Tuske S, Patel J, Jansen R et al..  2008.  The RNA polymerase "switch region" is a target for inhibitors.. Cell. 135(2):295-307. Abstract
The alpha-pyrone antibiotic myxopyronin (Myx) inhibits bacterial RNA polymerase (RNAP). Here, through a combination of genetic, biochemical, and structural approaches, we show that Myx interacts with the RNAP "switch region"--the hinge that mediates opening and closing of the RNAP active center cleft--to prevent interaction of RNAP with promoter DNA. We define the contacts between Myx and RNAP and the effects of Myx on RNAP conformation and propose that Myx functions by interfering with opening of the RNAP active-center cleft during transcription initiation. We further show that the structurally related alpha-pyrone antibiotic corallopyronin (Cor) and the structurally unrelated macrocyclic-lactone antibiotic ripostatin (Rip) function analogously to Myx. The RNAP switch region is distant from targets of previously characterized RNAP inhibitors, and, correspondingly, Myx, Cor, and Rip do not exhibit crossresistance with previously characterized RNAP inhibitors. The RNAP switch region is an attractive target for identification of new broad-spectrum antibacterial therapeutic agents.
Kim, Y, Ebright YW, Goodman AR, Reinberg D, Ebright RH.  2008.  Nonradioactive, ultrasensitive site-specific protein-protein photocrosslinking: interactions of alpha-helix 2 of TATA-binding protein with general transcription factor TFIIA and transcriptional repressor NC2.. Nucleic acids research. 36(19):6143-54. Abstract
We have developed an approach that enables nonradioactive, ultrasensitive (attamole sensitivity) site-specific protein-protein photocrosslinking, and we have applied the approach to the analysis of interactions of alpha-helix 2 (H2) of human TATA-element binding protein (TBP) with general transcription factor TFIIA and transcriptional repressor NC2. We have found that TBP H2 can be crosslinked to TFIIA in the TFIIA-TBP-DNA complex and in higher order transcription-initiation complexes, and we have mapped the crosslink to the 'connector' region of the TFIIA alpha/beta subunit (TFIIAalpha/beta). We further have found that TBP H2 can be crosslinked to NC2 in the NC2-TBP-DNA complex, and we have mapped the crosslink to the C-terminal 'tail' of the NC2 alpha-subunit (NC2alpha). Interactions of TBP H2 with the TFIIAalpha/beta connector and the NC2alpha C-terminal tail were not observed in crystal structures of TFIIA-TBP-DNA and NC2-TBP-DNA complexes, since relevant segments of TFIIA and NC2 were not present in truncated TFIIA and NC2 derivatives used for crystallization. We propose that interactions of TBP H2 with the TFIIAalpha/beta connector and the NC2alpha C-terminal tail provide an explanation for genetic results suggesting importance of TBP H2 in TBP-TFIIA interactions and TBP-NC2 interactions, and provide an explanation-steric exclusion-for competition between TFIIA and NC2.
Cellai, S, Mangiarotti L, Vannini N, Naryshkin N, Kortkhonjia E, Ebright RH, Rivetti C.  2007.  Upstream promoter sequences and alphaCTD mediate stable DNA wrapping within the RNA polymerase-promoter open complex.. EMBO reports. 8(3):271-8. Abstract
We show that the extent of stable DNA wrapping by Escherichia coli RNA polymerase (RNAP) in the RNAP-promoter open complex depends on the sequence of the promoter and, in particular, on the sequence of the upstream region of the promoter. We further show that the extent of stable DNA wrapping depends on the presence of the RNAP alpha-subunit carboxy-terminal domain and on the presence and length of the RNAP alpha-subunit interdomain linker. Our results indicate that the extensive stable DNA wrapping observed previously in the RNAP-promoter open complex at the lambda P(R) promoter is not a general feature of RNAP-promoter open complexes.
Popovych, N, Sun S, Ebright RH, Kalodimos CG.  2006.  Dynamically driven protein allostery.. Nature structural & molecular biology. 13(9):831-8. Abstract
Allosteric interactions are typically considered to proceed through a series of discrete changes in bonding interactions that alter the protein conformation. Here we show that allostery can be mediated exclusively by transmitted changes in protein motions. We have characterized the negatively cooperative binding of cAMP to the dimeric catabolite activator protein (CAP) at discrete conformational states. Binding of the first cAMP to one subunit of a CAP dimer has no effect on the conformation of the other subunit. The dynamics of the system, however, are modulated in a distinct way by the sequential ligand binding process, with the first cAMP partially enhancing and the second cAMP completely quenching protein motions. As a result, the second cAMP binding incurs a pronounced conformational entropic penalty that is entirely responsible for the observed cooperativity. The results provide strong support for the existence of purely dynamics-driven allostery.
Revyakin, A, Liu C, Ebright RH, Strick TR.  2006.  Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching.. Science (New York, N.Y.). 314(5802):1139-43. Abstract
Using single-molecule DNA nanomanipulation, we show that abortive initiation involves DNA "scrunching"--in which RNA polymerase (RNAP) remains stationary and unwinds and pulls downstream DNA into itself--and that scrunching requires RNA synthesis and depends on RNA length. We show further that promoter escape involves scrunching, and that scrunching occurs in most or all instances of promoter escape. Our results support the existence of an obligatory stressed intermediate, with approximately one turn of additional DNA unwinding, in escape and are consistent with the proposal that stress in this intermediate provides the driving force to break RNAP-promoter and RNAP-initiation-factor interactions in escape.
Kapanidis, AN, Margeat E, Ho S O, Kortkhonjia E, Weiss S, Ebright RH.  2006.  Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism.. Science (New York, N.Y.). 314(5802):1144-7. Abstract
Using fluorescence resonance energy transfer to monitor distances within single molecules of abortively initiating transcription initiation complexes, we show that initial transcription proceeds through a "scrunching" mechanism, in which RNA polymerase (RNAP) remains fixed on promoter DNA and pulls downstream DNA into itself and past its active center. We show further that putative alternative mechanisms for RNAP active-center translocation in initial transcription, involving "transient excursions" of RNAP relative to DNA or "inchworming" of RNAP relative to DNA, do not occur. The results support a model in which a stressed intermediate, with DNA-unwinding stress and DNA-compaction stress, is formed during initial transcription, and in which accumulated stress is used to drive breakage of interactions between RNAP and promoter DNA and between RNAP and initiation factors during promoter escape.
Tadigotla, VR, O Maoiléidigh D, Sengupta AM, Epshtein V, Ebright RH, Nudler E, Ruckenstein AE.  2006.  Thermodynamic and kinetic modeling of transcriptional pausing.. Proceedings of the National Academy of Sciences of the United States of America. 103(12):4439-44. Abstract
We present a statistical mechanics approach for the prediction of backtracked pauses in bacterial transcription elongation derived from structural models of the transcription elongation complex (EC). Our algorithm is based on the thermodynamic stability of the EC along the DNA template calculated from the sequence-dependent free energy of DNA-DNA, DNA-RNA, and RNA-RNA base pairing associated with (i) the translocational and size fluctuations of the transcription bubble; (ii) changes in the associated DNA-RNA hybrid; and (iii) changes in the cotranscriptional RNA secondary structure upstream of the RNA exit channel. The calculations involve no adjustable parameters except for a cutoff used to discriminate paused from nonpaused complexes. When applied to 100 experimental pauses in transcription elongation by Escherichia coli RNA polymerase on 10 DNA templates, the approach produces statistically significant results. We also present a kinetic model for the rate of recovery of backtracked paused complexes. A crucial ingredient of our model is the incorporation of kinetic barriers to backtracking resulting from steric clashes of EC with the cotranscriptionally generated RNA secondary structure, an aspect not included explicitly in previous attempts at modeling the transcription elongation process.
Napoli, AA, Lawson CL, Ebright RH, Berman HM.  2006.  Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: recognition of pyrimidine-purine and purine-purine steps.. Journal of molecular biology. 357(1):173-83. Abstract
The catabolite activator protein (CAP) bends DNA in the CAP-DNA complex, typically introducing 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, 5'-A1A2A3T4G5T6G7A8T9C10T11 -3' ("primary kink"). In previous work, we showed that CAP recognizes the nucleotide immediately 5' to the primary-kink site, T6, through an "indirect-readout" mechanism involving sequence effects on energetics of primary-kink formation. Here, to understand further this example of indirect readout, we have determined crystal structures of CAP-DNA complexes containing each possible nucleotide at position 6. The structures show that CAP can introduce a DNA kink at the primary-kink site with any nucleotide at position 6. The DNA kink is sharp with the consensus pyrimidine-purine step T6G7 and the non-consensus pyrimidine-purine step C6G7 (roll angles of approximately 42 degrees, twist angles of approximately 16 degrees ), but is much less sharp with the non-consensus purine-purine steps A6G7 and G6G7 (roll angles of approximately 20 degrees, twist angles of approximately 17 degrees). We infer that CAP discriminates between consensus and non-consensus pyrimidine-purine steps at positions 6-7 solely based on differences in the energetics of DNA deformation, but that CAP discriminates between the consensus pyrimidine-purine step and non-consensus purine-purine steps at positions 6-7 both based on differences in the energetics of DNA deformation and based on qualitative differences in DNA deformation. The structures further show that CAP can achieve a similar, approximately 46 degrees per DNA half-site, overall DNA bend through a sharp DNA kink, a less sharp DNA kink, or a smooth DNA bend. Analysis of these and other crystal structures of CAP-DNA complexes indicates that there is a large, approximately 28 degrees per DNA half-site, out-of-plane component of CAP-induced DNA bending in structures not constrained by end-to-end DNA lattice interactions and that lattice contacts involving CAP tend to involve residues in or near biologically functional surfaces.
Margeat, E, Kapanidis AN, Tinnefeld P, Wang Y, Mukhopadhyay J, Ebright RH, Weiss S.  2006.  Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes.. Biophysical journal. 90(4):1419-31. Abstract
Using total-internal-reflection fluorescence microscopy equipped with alternating-laser excitation, we were able to detect abortive initiation and promoter escape within single immobilized transcription complexes. Our approach uses fluorescence resonance energy transfer to monitor distances between a fluorescent probe incorporated in RNA polymerase (RNAP) and a fluorescent probe incorporated in DNA. We observe small, but reproducible and abortive-product-length-dependent, decreases in distance between the RNAP leading edge and DNA downstream of RNAP upon abortive initiation, and we observe large decreases in distance upon promoter escape. Inspection of population distributions and single-molecule time traces for abortive initiation indicates that, at a consensus promoter, at saturating ribonucleoside triphosphate concentrations, abortive-product release is rate-limiting (i.e., abortive-product synthesis and RNAP-active-center forward translocation are fast, whereas abortive-product dissociation and RNAP-active-center reverse translocation are slow). The results obtained using this new methodology confirm and extend those obtained from diffusing single molecules, and pave the way for real-time, single-molecule observations of the transitions between various states of the transcription complex throughout transcription.
Vrentas, CE, Gaal T, Ross W, Ebright RH, Gourse RL.  2005.  Response of RNA polymerase to ppGpp: requirement for the omega subunit and relief of this requirement by DksA.. Genes & development. 19(19):2378-87. Abstract
Previous studies have come to conflicting conclusions about the requirement for the omega subunit of RNA polymerase in bacterial transcription regulation. We demonstrate here that purified RNAP lacking omega does not respond in vitro to the effector of the stringent response, ppGpp. DksA, a transcription factor that works in concert with ppGpp to regulate rRNA expression in vivo and in vitro, fully rescues the ppGpp-unresponsiveness of RNAP lacking omega, likely explaining why strains lacking omega display a stringent response in vivo. These results demonstrate that omega plays a role in RNAP function (in addition to its previously reported role in RNAP assembly) and highlight the importance of inclusion of omega in RNAP purification protocols. Furthermore, these results suggest that either one or both of two short segments in the beta' subunit that physically link omega to the ppGpp-binding region of the enzyme may play crucial roles in ppGpp and DksA function.
Kapanidis, AN, Margeat E, Laurence TA, Doose S, Ho S O, Mukhopadhyay J, Kortkhonjia E, Mekler V, Ebright RH, Weiss S.  2005.  Retention of transcription initiation factor sigma70 in transcription elongation: single-molecule analysis.. Molecular cell. 20(3):347-56. Abstract
We report a single-molecule assay that defines, simultaneously, the translocational position of a protein complex relative to DNA and the subunit stoichiometry of the complex. We applied the assay to define translocational positions and sigma70 contents of bacterial transcription elongation complexes in vitro. The results confirm ensemble results indicating that a large fraction, approximately 70%-90%, of early elongation complexes retain sigma70 and that a determinant for sigma70 recognition in the initial transcribed region increases sigma70 retention in early elongation complexes. The results establish that a significant fraction, approximately 50%-60%, of mature elongation complexes retain sigma70 and that a determinant for sigma70 recognition in the initial transcribed region does not appreciably affect sigma70 retention in mature elongation complexes. The results further establish that, in mature elongation complexes that retain sigma70, the half-life of sigma70 retention is long relative to the time-scale of elongation, suggesting that some complexes may retain sigma70 throughout elongation.
Altman, S, Bassler BL, Beckwith J, Belfort M, Berg HC, Bloom B, Brenchley JE, Campbell A, Collier JR, Connell N et al..  2005.  An open letter to Elias Zerhouni.. Science (New York, N.Y.). 307(5714):1409-10.
Nickels, BE, Garrity SJ, Mekler V, Minakhin L, Severinov K, Ebright RH, Hochschild A.  2005.  The interaction between sigma70 and the beta-flap of Escherichia coli RNA polymerase inhibits extension of nascent RNA during early elongation.. Proceedings of the National Academy of Sciences of the United States of America. 102(12):4488-93. Abstract
The sigma-subunit of bacterial RNA polymerase (RNAP) is required for promoter-specific transcription initiation. This function depends on specific intersubunit interactions that occur when sigma associates with the RNAP core enzyme to form RNAP holoenzyme. Among these interactions, that between conserved region 4 of sigma and the flap domain of the RNAP beta-subunit (beta-flap) is critical for recognition of the major class of bacterial promoters. Here, we describe the isolation of amino acid substitutions in region 4 of Escherichia coli sigma(70) that have specific effects on the sigma(70) region 4/beta-flap interaction, either weakening or strengthening it. Using these sigma(70) mutants, we demonstrate that the sigma region 4/beta-flap interaction also can affect events occurring downstream of transcription initiation during early elongation. Specifically, our results provide support for a structure-based proposal that, when bound to the beta-flap, sigma region 4 presents a barrier to the extension of the nascent RNA as it emerges from the RNA exit channel. Our findings support the view that the transition from initiation to elongation involves a staged disruption of sigma-core interactions.
Knight, JL, Mekler V, Mukhopadhyay J, Ebright RH, Levy RM.  2005.  Distance-restrained docking of rifampicin and rifamycin SV to RNA polymerase using systematic FRET measurements: developing benchmarks of model quality and reliability.. Biophysical journal. 88(2):925-38. Abstract
We are developing distance-restrained docking strategies for modeling macromolecular complexes that combine available high-resolution structures of the components and intercomponent distance restraints derived from systematic fluorescence resonance energy transfer (FRET) measurements. In this article, we consider the problem of docking small-molecule ligands within macromolecular complexes. Using simulated FRET data, we have generated a series of benchmarks that permit estimation of model accuracy based on the quantity and quality of FRET-derived distance restraints, including the number, random error, systematic error, distance distribution, and radial distribution of FRET-derived distance restraints. We find that expected model accuracy is 10 A or better for models based on: i), > or =20 restraints with up to 15% random error and no systematic error, or ii), > or =20 restraints with up to 15% random error, up to 10% systematic error, and a symmetric radial distribution of restraints. Model accuracies can be improved to 5 A or better by increasing the number of restraints to > or =40 and/or by optimizing the distance distribution of restraints. Using experimental FRET data, we have defined the positions of the binding sites within bacterial RNA polymerase of the small-molecule inhibitors rifampicin (Rif) and rifamycin SV (Rif SV). The inferred binding sites for Rif and Rif SV were located with accuracies of, respectively, 7 and 10 A relative to the crystallographically defined binding site for Rif. These accuracies agree with expectations from the benchmark simulations and suffice to indicate that the binding sites for Rif and Rif SV are located within the RNA polymerase active-center cleft, overlapping the binding site for the RNA-DNA hybrid.
Revyakin, A, Ebright RH, Strick TR.  2005.  Single-molecule DNA nanomanipulation: improved resolution through use of shorter DNA fragments.. Nature methods. 2(2):127-38.