Structural Basis of Transcription Inhibition by Fidaxomicin (Lipiarmycin A3).

Fidaxomicin is an antibacterial drug in clinical use for treatment of Clostridium difficile diarrhea. The active ingredient of fidaxomicin, lipiarmycin A3 (Lpm), functions by inhibiting bacterial RNA polymerase (RNAP). Here we report a cryo-EM structure of Mycobacterium tuberculosis RNAP holoenzyme in complex with Lpm at 3.5-Å resolution. The structure shows that Lpm binds at the base of the RNAP "clamp." The structure exhibits an open conformation of the RNAP clamp, suggesting that Lpm traps an open-clamp state. Single-molecule fluorescence resonance energy transfer experiments confirm that Lpm traps an open-clamp state and define effects of Lpm on clamp dynamics. We suggest that Lpm inhibits transcription by trapping an open-clamp state, preventing simultaneous interaction with promoter -10 and -35 elements. The results account for the absence of cross-resistance between Lpm and other RNAP inhibitors, account for structure-activity relationships of Lpm derivatives, and enable structure-based design of improved Lpm derivatives.

Structural Basis of Mycobacterium tuberculosis Transcription and Transcription Inhibition.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which kills 1.8 million annually. Mtb RNA polymerase (RNAP) is the target of the first-line antituberculosis drug rifampin (Rif). We report crystal structures of Mtb RNAP, alone and in complex with Rif, at 3.8-4.4 Å resolution. The results identify an Mtb-specific structural module of Mtb RNAP and establish that Rif functions by a steric-occlusion mechanism that prevents extension of RNA. We also report non-Rif-related compounds-Nα-aroyl-N-aryl-phenylalaninamides (AAPs)-that potently and selectively inhibit Mtb RNAP and Mtb growth, and we report crystal structures of Mtb RNAP in complex with AAPs. AAPs bind to a different site on Mtb RNAP than Rif, exhibit no cross-resistance with Rif, function additively when co-administered with Rif, and suppress resistance emergence when co-administered with Rif.

Structural Basis of Transcription Inhibition by CBR Hydroxamidines and CBR Pyrazoles.

CBR hydroxamidines are small-molecule inhibitors of bacterial RNA polymerase (RNAP) discovered through high-throughput screening of synthetic-compound libraries. CBR pyrazoles are structurally related RNAP inhibitors discovered through scaffold hopping from CBR hydroxamidines. CBR hydroxamidines and pyrazoles selectively inhibit Gram-negative bacterial RNAP and exhibit selective antibacterial activity against Gram-negative bacteria. Here, we report crystal structures of the prototype CBR hydroxamidine, CBR703, and a CBR pyrazole in complex with E. coli RNAP holoenzyme. In addition, we define the full resistance determinant for CBR703, show that the binding site and resistance determinant for CBR703 do not overlap the binding sites and resistance determinants of other characterized RNAP inhibitors, show that CBR703 exhibits no or minimal cross-resistance with other characterized RNAP inhibitors, and show that co-administration of CBR703 with other RNAP inhibitors results in additive antibacterial activities. The results set the stage for structure-based optimization of CBR inhibitors as antibacterial drugs.

Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation.

A three-step procedure comprising (1) unnatural-amino-acid mutagenesis with 4-azido-phenylalanine, (2) Staudinger-Bertozzi ligation with a probe-phosphine derivative, and (3) in vitro reconstitution of RNA polymerase (RNAP) enables the efficient site-specific incorporation of a fluorescent probe, a spin label, a cross-linking agent, a cleaving agent, an affinity tag, or any other biochemical or biophysical probe, at any site of interest in RNAP. Straightforward extensions of the procedure enable the efficient site-specific incorporation of two or more different probes in two or more different subunits of RNAP. We present protocols for synthesis of probe-phosphine derivatives, preparation of RNAP subunits and the transcription initiation factor σ, unnatural amino acid mutagenesis of RNAP subunits and σ, Staudinger ligation with unnatural-amino-acid-containing RNAP subunits and σ, quantitation of labelling efficiency and labelling specificity, and reconstitution of RNAP.

Transcription inhibition by the depsipeptide antibiotic salinamide A.

We report that bacterial RNA polymerase (RNAP) is the functional cellular target of the depsipeptide antibiotic salinamide A (Sal), and we report that Sal inhibits RNAP through a novel binding site and mechanism. We show that Sal inhibits RNA synthesis in cells and that mutations that confer Sal-resistance map to RNAP genes. We show that Sal interacts with the RNAP active-center 'bridge-helix cap' comprising the 'bridge-helix N-terminal hinge', 'F-loop', and 'link region'. We show that Sal inhibits nucleotide addition in transcription initiation and elongation. We present a crystal structure that defines interactions between Sal and RNAP and effects of Sal on RNAP conformation. We propose that Sal functions by binding to the RNAP bridge-helix cap and preventing conformational changes of the bridge-helix N-terminal hinge necessary for nucleotide addition. The results provide a target for antibacterial drug discovery and a reagent to probe conformation and function of the bridge-helix N-terminal hinge.DOI: http://dx.doi.org/10.7554/eLife.02451.001.

GE23077 binds to the RNA polymerase 'i' and 'i+1' sites and prevents the binding of initiating nucleotides.

Using a combination of genetic, biochemical, and structural approaches, we show that the cyclic-peptide antibiotic GE23077 (GE) binds directly to the bacterial RNA polymerase (RNAP) active-center 'i' and 'i+1' nucleotide binding sites, preventing the binding of initiating nucleotides, and thereby preventing transcription initiation. The target-based resistance spectrum for GE is unusually small, reflecting the fact that the GE binding site on RNAP includes residues of the RNAP active center that cannot be substituted without loss of RNAP activity. The GE binding site on RNAP is different from the rifamycin binding site. Accordingly, GE and rifamycins do not exhibit cross-resistance, and GE and a rifamycin can bind simultaneously to RNAP. The GE binding site on RNAP is immediately adjacent to the rifamycin binding site. Accordingly, covalent linkage of GE to a rifamycin provides a bipartite inhibitor having very high potency and very low susceptibility to target-based resistance. DOI: http://dx.doi.org/10.7554/eLife.02450.001.

Frequency, spectrum, and nonzero fitness costs of resistance to myxopyronin in Staphylococcus aureus.

The antibiotic myxopyronin (Myx) functions by inhibiting bacterial RNA polymerase (RNAP). The binding site on RNAP for Myx-the RNAP "switch region SW1/SW2 subregion"-is different from the binding site on RNAP for the RNAP inhibitor currently used in broad-spectrum antibacterial therapy, rifampin (Rif). Here, we report the frequency, spectrum, and fitness costs of Myx resistance in Staphylococcus aureus. The resistance rate for Myx is 4 × 10(-8) to 7 × 10(-8) per generation, which is equal within error to the resistance rate for Rif (3 × 10(-8) to 10 × 10(-8) per generation). Substitutions conferring Myx resistance were obtained in the RNAP ß subunit [six substitutions: V1080(1275)I, V1080(1275)L, E1084(1279)K, D1101(1296)E, S1127(1322)L, and S1127(1322)P] and the RNAP ß' subunit [five substitutions: K334(345)N, T925(917)K, T925(917)R, G1172(1354)C, and G1172(1354)D] (residues numbered as in Staphylococcus aureus RNAP and, in parentheses, as in Escherichia coli RNAP). Sites of substitutions conferring Myx resistance map to the RNAP switch region SW1/SW2 subregion and do not overlap the binding site on RNAP for Rif, and, correspondingly, Myx-resistant mutants exhibit no cross-resistance to Rif. All substitutions conferring Myx resistance exhibit significant fitness costs (4 to 15% per generation). In contrast, at least three substitutions conferring Rif resistance exhibit no fitness costs (≤0% per generation). The observation that all Myx-resistant mutants have significant fitness costs whereas at least three Rif-resistant mutants have no fitness costs, together with the previously established inverse correlation between fitness cost and clinical prevalence, suggests that Myx resistance is likely to have lower clinical prevalence than Rif resistance.

Opening and closing of the bacterial RNA polymerase clamp.

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.

New target for inhibition of bacterial RNA polymerase: 'switch region'.

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.

Azide-specific labeling of biomolecules by Staudinger-Bertozzi ligation phosphine derivatives of fluorescent probes suitable for single-molecule fluorescence spectroscopy.

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.

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.

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.

HEN1 recognizes 21-24 nt small RNA duplexes and deposits a methyl group onto the 2' OH of the 3' terminal nucleotide.

microRNAs (miRNAs) and small interfering RNAs (siRNAs) in plants bear a methyl group on the ribose of the 3' terminal nucleotide. We showed previously that the methylation of miRNAs and siRNAs requires the protein HEN1 in vivo and that purified HEN1 protein methylates miRNA/miRNA* duplexes in vitro. In this study, we show that HEN1 methylates both miRNA/miRNA* and siRNA/siRNA* duplexes in vitro with a preference for 21-24 nt RNA duplexes with 2 nt overhangs. We also demonstrate that HEN1 deposits the methyl group on to the 2' OH of the 3' terminal nucleotide. Among various modifications that can occur on the ribose of the terminal nucleotide, such as 2'-deoxy, 3'-deoxy, 2'-O-methyl and 3'-O-methyl, only 2'-O-methyl on a small RNA inhibits the activity of yeast poly(A) polymerase (PAP). These findings indicate that HEN1 specifically methylates miRNAs and siRNAs and implicate the importance of the 2'-O-methyl group in the biology of RNA silencing.

Structural basis of transcription activation: the CAP-alpha CTD-DNA complex.

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.

Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex.

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.