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


Introduction
Salinamide A (Sal; SalA) and salinamide B (SalB) are structurally related bicyclic depsipeptide antibiotics, each consisting of seven amino acids and two non-amino-acid residues (Trischman et al., 1994;Moore et al., 1999;Figure 1A). SalA and SalB are produced by Streptomyces sp. CNB-091, a marine bacterium isolated from the surface of the jellyfish Cassiopeia xamachana (Trischman et al., 1994;Moore and Seng, 1998;Moore et al., 1999), and SalA also is produced by Streptomyces sp. NRRL 21611, a soil bacterium (Miao et al., 1997). SalA and SalB exhibit antibacterial activity against both Gram-positive and Gram-negative bacterial pathogens, particularly Enterobacter cloacae and Haemophilus influenzae, but do not exhibit cytotoxicity against mammalian cells (Trischman et al., 1994;Moore et al., 1999;Figure 1B). SalA and SalB inhibit both Gram-positive and Gram-negative bacterial RNA polymerase (RNAP) in vitro, but do not inhibit human RNAP I, II, or III in vitro (Miao et al., 1997;Figure 1C). A total synthesis of SalA has been reported (Tan and Ma, 2008).
Although previous work had established that Sal exhibits RNAP-inhibitory activity in a purified system in vitro and antibacterial activity in culture (Trischman et al., 1994;Miao et al., 1997;Moore et al., 1999), previous work had not established a causal relationship between the RNAP-inhibitory activity of Sal and the antibacterial activity of Sal (i.e., had not established that RNAP is the functional cellular target of Sal). In addition, previous work had not provided information regarding the binding site, mechanism, and structural basis of inhibition of RNAP by Sal.
In this work, we show that RNAP is the functional cellular target of Sal, we show that Sal inhibits RNAP through a novel binding site and novel mechanism, we determine crystal structures that define

Sal inhibits RNAP in cells
As a first step to determine whether the RNAP-inhibitory activity of Sal is responsible for the antibacterial activity of Sal in culture, we assessed whether Sal inhibits RNAP in bacterial cells in culture. To do this, we assayed macromolecular synthesis by bacterial cells in culture, monitoring incorporation of [ 14 C]-thymidine into DNA, [ 14 C]-uracil into RNA, and [ 14 C]-amino acids into protein. The results in Figure 2A shows that addition of Sal to cultures inhibits RNA synthesis at the first time point following addition and inhibits protein synthesis at later time points. Addition of Sal has no effect on DNA synthesis. The pattern observed for Sal matches the pattern observed for the reference RNAP inhibitor rifampin (Rif; compare red lines and blue lines in Figure 2A; Lancini and Sartori, 1968;Lancini et al., 1969), and matches the pattern expected from first principles for an RNAP inhibitor (i.e., immediate inhibition of RNAP-dependent RNA synthesis and later inhibition of RNA-dependent protein synthesis; Sergio et al., 1975;Irschik et al., 1983Irschik et al., , 1985Irschik et al., , 1995. We conclude that Sal inhibits RNA synthesis in bacterial cells in culture, and we infer that Sal inhibits RNAP in bacterial cells in culture.

Sal-resistant mutations occur in RNAP subunit genes
As a second step to determine whether the RNAP-inhibitory activity of Sal is responsible for the antibacterial activity of Sal, we assessed whether Sal-resistant mutations occur in RNAP subunit genes. To do this, we isolated spontaneous Sal-resistant mutants and then PCR-amplified and sequenced genes for RNAP subunits ( Figure 2B,C).
Spontaneous Sal-resistant mutants were isolated by plating E. coli strain, D21f2tolC-a strain with cell-envelope defects resulting in increased uptake and decreased efflux of small molecules, including Sal (Fralick and Burns-Keliher, 1994; DD and RHE, unpublished)-on agar containing Sal and identifying Sal-resistant colonies. For each Sal-resistant isolate, genomic DNA was prepared and the genes for the largest and second-largest RNAP subunits, rpoC encoding RNAP β′ subunit and rpoB encoding RNAP β subunit, were PCR-amplified and sequenced. Spontaneous Sal-resistant mutants were isolated eLife digest The need for new antibiotics is becoming increasingly critical, as more and more bacteria become resistant to existing drugs. To develop new treatments, researchers need to understand how antibiotics work. One way antibiotics can kill bacteria is by targeting an enzyme called bacterial RNA polymerase. This enzyme builds chains of RNA that bacteria need to survive.
Sal is an antibiotic produced by a marine bacterium found on the surface of a species of jellyfish. Degen, Feng et al. show that Sal kills bacteria by inhibiting bacterial RNA polymerase and explain how Sal inhibits RNA polymerase. Sal binds to a rod-like structural element within RNA polymerase known as the 'bridge helix'. The bridge helix has been proposed by others to contain two 'hinges' that open and close-allowing the bridge helix to bend and unbend-at specific steps in the cycle through which RNA polymerase builds an RNA chain. Degen, Feng et al. show that Sal binds directly to one of the two hinges and show that Sal binds to the hinge in the unbent state. Therefore, Degen, Feng et al. propose that Sal inhibits the enzyme by preventing the hinge from bending.
The binding site on RNA polymerase for Sal is different from, and does not overlap, the binding sites of current antibacterial drugs. As a result, Sal is able to kill bacteria that are resistant to current antibacterial drugs. When Degen, Feng et al. administered Sal in combination with a current antibacterial drug that targets RNA polymerase, bacteria did not detectably develop resistance to either Sal or the current antibacterial drug.
The structure of the complex between Sal and RNA polymerase suggests several ways that Sal could be modified to improve its ability to interact with RNA polymerase, thereby potentially increasing Sal's antibacterial activity. Future research could develop a range of new drugs based on Sal that could kill bacteria more effectively. (2 x MIC). Blue, Rif (2 x MIC). Asterisks, statistically significant differences between no-inhibitor data and Sal data (t test; p<0.01). (B and C) Sal-resistant mutations occur in RNAP subunit genes. MIC wild-type,SalA = 0.049 µg/ml; MIC wild−type,SalB = 0.20 µg/ml. DOI: 10.7554/eLife.02451.004 with a frequency of ∼1 × 10 −9 ( Figure 2B). A total of 47 independent Sal-resistant mutants were isolated, PCR-amplified, and sequenced ( Figure 2B). Strikingly, 100% (47/47) of the analyzed Sal-resistant mutants were found to contain mutations in genes for RNAP subunits: 36 were found to contain mutations in rpoC and 11 were found to contain mutations in rpoB ( Figure 2B).
In parallel work, we isolated and sequenced induced Sal-resistant mutants (Supplementary file 1). Random mutagenesis of plasmid-borne rpoC and rpoB genes was performed using error-prone PCR, mutagenized plasmid DNA was introduced into E. coli strain D21f2tolC by transformation, transformants were plated on media containing Sal, and Sal-resistant clones were isolated. The plasmid-borne, induced Sal-resistant mutants were found to contain mutations in the same rpoC and rpoB segments as the spontaneous Sal-resistant mutants (compare Supplementary file 1 and Figure 2C). Transfer of plasmids carrying plasmid-borne, induced Sal-resistant mutants was found to transfer the Sal-resistant phenotype, indicating that no mutation outside of rpoC or rpoB is required for Sal-resistance.
From the analysis of spontaneous and induced Sal-resistant mutants, we conclude that a single substitution in an RNAP subunit gene, either rpoC or rpoB, is sufficient to confer Sal-resistance, and we infer that RNAP is the functional cellular target for Sal.

Sal-resistant mutations define the Sal target
In the three-dimensional structure of RNAP, the sites of substitutions conferring Sal-resistance form a tight cluster ('the Sal target'; green surface in Figure 3A). The dimensions of the Sal target are ∼35 Å × ∼18 Å × ∼12 Å. The Sal target is sufficiently large to be able to encompass Sal (∼16 Å × ∼12 Å × ∼10 Å). Based on the observation that substitutions of the Sal target result in Sal-resistance ( Figure 3A), we infer that the Sal target is the binding site for Sal on RNAP.

The Sal target overlaps the RNAP active-center region
The Sal target is located adjacent to, and partly overlaps, the RNAP active-center region ( Figure 3A). We infer that Sal likely inhibits RNAP by inhibiting RNAP active-center function.
Mapping of substitutions conferring Sal-resistance onto the three-dimensional structure of a transcription elongation complex comprising RNAP, DNA, RNA, and an NTP (Vassylyev et al., 2007b) indicates that the Sal target does not overlap the RNAP active-center catalytic Mg 2+ ion and does not overlap the RNAP residues that interact with the DNA template, the RNA product, and the NTP substrate. We infer that Sal likely inhibits RNAP active-center function allosterically, through effects on RNAP conformation, and not through direct, steric interactions with the RNAP residues that mediate bond formation, template binding, product binding, or substrate binding.

The Sal target overlaps the RNAP active-center bridge-helix cap
The Sal target overlaps an RNAP active-center module referred to as the 'bridge-helix cap', which, in turn, comprises three active-center subregions: the 'bridge-helix N-terminal hinge' (BH-H N ), the 'F-loop', and the 'link region' ( Figure 3B; active-center subregion nomenclature as in Weinzierl 2010 and. 18 of the 21 identified substitutions conferring Sal-resistance, and all substitutions conferring high-level Sal-resistance, map to these RNAP active-center subregions ( Figure 3B). It recently has been proposed that the BH-H N undergoes conformational changes coupled to, and essential for, the nucleotide-addition cycle in transcription initiation and transcription elongation, and that the F-loop, and possibly the link region, control these conformational changes (Hein and Landick, 2010;Weinzierl, 2010;Kireeva et al., 2012;Nedialkov et al., 2013). Specifically, it has been proposed that the BH-H N segment comprising β′ residues 779-783-a segment that includes the sites of 6 of the 21 identified substitutions conferring Sal-resistance, and 4 of the 9 substitutions conferring high-level Sal-resistance-undergoes a hinge-opening/hinge-closing conformational cycle coupled to the nucleotide-addition cycle (Hein and Landick, 2010;Weinzierl, 2010;Kireeva et al., 2012;Nedialkov et al., 2013). (These proposals are supported by results of mutagenesis studies and molecular-dynamics simulations. However, these proposals have not been definitively established. Crystal structures showing an 'open' (unbent) BH-H N conformational state have been reported, but a crystal structure showing a 'closed' (bent) BH-H N conformational state has not been reported.) Based on the strong, nearly one-for-one, correspondence between the Sal target and the active-center subregions proposed to mediate and control the BH-H N hinge-opening/hinge-closing conformational cycle, we suggest that Sal inhibits RNAP active-center function by inhibiting the proposed BH-H N hinge-opening and/or hinge-closing.
Sal does not exhibit cross-resistance with the RNAP inhibitors rifampin, streptolydigin, CBR703, myxopyronin, and lipiarmycin Consistent with the absence of overlap between the Sal target and the Rif, Stl, CBR703, Myx, and Lpm targets, Sal-resistant mutants do not exhibit cross-resistance with Rif, Stl, CBR703, Myx, and Lpm ( Figure 4B). Conversely, Rif-resistant, Stl-resistant, CBR703-resistant, Myx-resistant, and Lpm-resistant mutants do not exhibit cross-resistance with Sal ( Figure 4C).   Mukhopadhyay et al., 2008), showing sites of substitutions that confer resistance to Sal (green; Figures 2, 3), Rif (red;Ovchinnikov et al., 1981Ovchinnikov et al., , 1983Lisitsyn et al., 1984;Jin and Gross, 1988; For approximately one-quarter to one-half of Sal-resistant substitutions, not only is there no crossresistance to Stl and CBR703, but also there is significant (≥ fourfold) hyper-susceptibility to Stl and CBR703 (data in blue in Figure 4B). Resistance to a first inhibitor of an enzyme and hyper-susceptibility to a second inhibitor of the enzyme generally is understood to indicate that the two inhibitors affect different reaction steps of the enzyme and/or bind to and stabilize different conformational states of the enzyme (Tachedjian et al., 1996;Selmi et al., 2003). We infer that Sal may inhibit a different RNAP reaction step than Stl and CBR703 and/or may bind to and stabilize a different RNAP conformational state than Stl and CBR703.

Co-administration of Sal with rifampin or myxopyronin suppresses the emergence of resistance
The absence of overlap between the Sal target and other RNAP inhibitor targets, and the absence of cross-resistance between Sal and other RNAP inhibitors, suggests that the co-administration of Sal and another RNAP inhibitor may result in an extremely low, effectively undetectable, spontaneous resistance rate, representing the product of the spontaneous resistance rate for Sal and the spontaneous resistance rate for the other RNAP inhibitor. (For many pairs of antibacterial agents having different targets and no cross-resistance, co-administration potentially results in a spontaneous resistance rate comparable to the product of the individual spontaneous resistance rates [Fischbach, 2011]. This is true even for pairs of antibacterial agents that function through the same pathway and same target protein [Fischbach, 2011].) The results in Figure 4D support this hypothesis. Thus, co-administration of Sal (resistance rate = 2 × 10 −9 per generation) and Rif (resistance rate = 1 × 10 −9 per generation) results in a resistance rate below the limit of detection (<2 × 10 −12 per generation). In the same manner, co-administration of Sal (resistance rate 2 × 10 −9 per generation) and Myx (resistance rate 3 × 10 −10 per generation) results in a resistance rate below the limit of detection (<2 × 10 −12 per generation). The observed suppression of the emergence of spontaneous resistance has practical implications, in view of the fact that susceptibility to spontaneous resistance is the main limiting factor in clinical use of Rif (Floss and Yu, 2005) and has been cited as a potential barrier to clinical use of Myx (Moy et al., 2011).

Sal does not inhibit formation of a transcription initiation complex
To define the mechanistic basis of transcription inhibition by Sal, we assessed the effects of Sal on individual reaction steps in transcription initiation and transcription elongation ( Figure 5).
The results in Figure 5A show that Sal does not inhibit formation of a heparin-resistant RNAPpromoter open complex. The results indicate that the mechanism of transcription inhibition by Sal differs from the mechanisms of transcription inhibition by Myx and Lpm, both of which inhibit the formation of the RNAP-promoter open complex (Mukhopadhyay et al., 2008;Belogurov et al., 2009;Tupin et al., 2010;Srivastava et al., 2011).

Sal inhibits nucleotide addition in transcription elongation
The results in Figure 5C show that Sal also inhibits nucleotide addition in transcription elongation. In transcription elongation, Sal inhibits nucleotide addition both at non-pause sites ( Figure 5C) and at type-I and type-II pause sites (hairpin-stabilized pauses and backtrackingstabilized pauses; Figure 5-figure supplement 2) and inhibits not only the forward reaction of nucleotide addition but also the reverse reaction, pyrophosphorolysis ( Figure 5-figure supplement 3). The results confirm that the mechanism of transcription inhibition by Sal differs from the mechanisms of transcription inhibition by Rif, Myx, and Lpm, which do not inhibit transcription elongation ( Figure 5C; McClure and Cech, 1978;Mukhopadhyay et al., 2008;Belogurov et al., 2009;Tupin et al., 2010;Srivastava et al., 2011).

Sal inhibits nucleotide addition noncompetitively
The results in Figure 5D show that inhibition by Sal is noncompetitive with respect to NTP substrate. The K i for inhibition is 0.2 µM, which is equal to the IC50 for inhibition of transcription (compare Figures 5C and 1C). The results indicate that Sal does not inhibit the NTP binding sub-reaction of the nucleotide-addition cycle, but instead inhibits one or more of the bondformation, pyrophosphate-release, and translocation sub-reactions of the nucleotide-addition cycle.

Transcription inhibition by Sal does not require the RNAP trigger loop
The results in Figure 5E show that transcription inhibition by Sal does not require the RNAP activecenter subregion referred to as the 'trigger loop'. Thus, Sal inhibits wild-type RNAP and an RNAPderivative having a deletion of the trigger loop to the same extent and with nearly the same concentration-dependence. These results indicate that the mechanism of transcription inhibition by Sal differs from the mechanism of transcription inhibition by Stl, which absolutely requires the RNAP trigger loop (Temiakov et al., 2005).
Taken together, the results in Figure 5 establish that Sal inhibits RNAP through a mechanism different from the mechanisms of the previously  Figure 4B) suggests, but does not prove, that Sal inhibits RNAP through a mechanism that is also different from the mechanism of the previously characterized RNAP inhibitor CBR703. Based on the data presented to this point, we suggest that Sal inhibits RNAP through a novel binding site and a novel mechanism. Specifically, we suggest that Sal interacts with a binding site in the bridge-helix cap and allosterically interferes with the conformational dynamics of the BH-H N required for one or more of bond formation, pyrophosphate release, and translocation in the nucleotide-addition cycle of transcription initiation and transcription elongation. [At the time this work was performed, all published crystal structures of bacterial RNAP and bacterial RNAP complexes had employed RNAP from the genus Thermus. However, it was found that Sal did not inhibit RNAP from the genus Thermus ( Figure 1C). Therefore, it was necessary to determine both a reference crystal structure of a Salsusceptible bacterial RNAP and a crystal structure of the Sal-susceptible RNAP in complex with Sal.] Figure 6A shows the resulting crystal structure of E. coli RNAP holoenzyme at 3.9 Å resolution. In the structure, the conformations and interactions of RNAP β′ subunit, β subunit, α I subunit N-terminal domain (αNTD I ), α II subunit N-terminal domain (αNTD II ), ω subunit, and σ 70 regions 1.2-4 in our structure match those in recently published structures of E. coli RNAP holoenzyme (Murakami, 2013;Zuo et al., 2013;Bae et al., 2013). Our structure also includes the α I subunit C-terminal domain (αCTD I ), with a conformation and interactions matching those in the structure of Murakami, 2013 (Figure 6figure supplement 1). (αCTD I was not present in the RNAP derivatives used for crystallization in the structures of Zuo et al., 2013 andBae et al., 2013.) The structure also includes the α II subunit C-terminal domain (αCTD II ), positioned adjacent to, and in contact with, αNTD I , the β flap, and β dispensable region 2 (βDR2) (Figure 6-figure supplement 1). (αCTD II was not ordered in the structure of Murakami, 2013, and was not present in the RNAP derivatives used for crystallization in Zuo et al., 2013 andBae et al., 2013.) Figures 6B and C show the corresponding structure of E. coli RNAP holoenzyme in complex with Sal at 3.9 Å resolution. The structure shows unambiguous experimental electron density for Sal in the genetically-defined Sal target, confirming the hypothesis that the genetically-defined Sal target represents the binding site for Sal on RNAP ( Figure 6B,C).

Structural basis of transcription inhibition by Sal: crystal structure of E. coli RNAP holoenzyme in complex with a bromine-containing Sal derivative
To confirm the binding position and binding orientation of Sal shown in Figure 6B,C, we prepared a bromine-containing Sal derivative, and collected X-ray diffraction data for E. coli RNAP holoenzyme in complex with the bromine-containing Sal derivative (Figure 7; Supplementary file 3). The brominecontaining Sal-derivative ('Sal-Br') contained a residue-9 bromohydrin moiety structurally related to the residue-9 chlorohydrin moiety of SalB (compare Figures 7A and 1A). Sal-Br was prepared by semisynthesis from SalA, exploiting the unique chemical reactivity of the residue-9 epoxide moiety of SalA ( Figure 7A). Sal-Br was found to exhibit essentially full RNAP-inhibitory activity and essentially full antibacterial activity ( Figure 7B).
The RNAP-Sal-Br complex exhibited electron density for Sal-Br matching the electron density in the RNAP-Sal complex for Sal (blue mesh in Figures 6C and 7C) and exhibited a single peak of Br  anomalous difference density immediately adjacent to the electron density for Sal-Br, in the position expected for a Br atom covalently bonded to a carbon atom of the Sal-Br residue-9 bromohydrin (pink mesh in Figure 7C). The results unequivocally confirm the ligand binding position and ligand binding orientation.

Structural basis of transcription inhibition by Sal: Sal makes direct interactions with the RNAP bridge-helix cap
The structural information shows that Sal binds within the RNAP bridge-helix cap, making direct interactions with the BH-H N , the fork loop, and the link region ( Figures 6C, 7C, and 8). Sal makes direct interactions with all five residues at which substitutions conferring high-level (≥128-fold) Sal-resistance are obtained (β′ residues Arg738, Ala779, and Gly782, and β residues Asp675 and Asn677; red in Figure 8A). Substitution of β′ residue Arg738 would be expected to disrupt an H-bond between RNAP and Sal ( Figure 8B,C). Substitution of β′ residue Ala779 or Gly782 by any residue having a larger sidechain would be expected to introduce severe steric clash between RNAP and Sal ( Figure 8B,C). Substitution of β residues Asp675 and Asn677 would be expected to disrupt both H-bonds and van der Waals interactions between RNAP and Sal ( Figure 8B green) and sites of substitutions that confer high-level Sal-resistance (red). Views and labels as in Figures 6C and 7C. (B) Contacts between RNAP and Sal (stereoview). Gray, RNAP backbone (ribbon representation) and RNAP sidechain carbon atoms (stick representation). Green, Sal carbon atoms. Red, Figure 8. Continued on next page and the quality of electron density maps for residues of Sal and residues of RNAP close to Sal, the inferred proximities of individual residues of Sal to individual residues of RNAP are secure, but the inferred details of H-bonds and van der Waals interactions are, at least in part, provisional.) Six of the RNAP residues that make direct contact with Sal are conserved across Gram-positive bacterial RNAP, Gram-negative bacterial RNAP, and human RNAP I, II, and III (β′ residues 739,745,778,779,782,and 785;Figures 3B and 8B,C). Nine RNAP residues that contact Sal are conserved in Gram-positive bacterial RNAP and Gram-negative bacterial RNAP, but are not conserved, and indeed are radically different, in human RNAP I, II, and III (β′ residues 738, 744, 746, 747, 748, 775, and 781, and β residues 675 and 677; Figures 3B and 8B,C). The observed interactions account for, and explain, the observation that Sal inhibits Gram-positive and Gram-negative bacterial RNAP, but does not inhibit human RNAP I, II, and III ( Figure 1C).
Four of the five Sal-contacting residues in the RNAP BH-H N are conserved from bacterial RNAP to human RNAP (β′ residues 778, 779, 782, and 785), presumably reflecting constraints on sequence variation imposed by the functionally essential, conformationally dynamic, BH-H N . In contrast, only two of the nine Sal-contacting residues in the RNAP fork loop are conserved from bacterial RNAP to human RNAP (β′ residues 739 and 745), and no Sal-contacting residues in the RNAP link region are conserved from bacterial RNAP to human RNAP, presumably reflecting lower constraints on sequence variation in these RNAP regions. The pattern of residue conservation observed for Sal is reminiscent of the pattern of residue conservation observed for the RNAP inhibitor Myx (Mukhopadhyay et al., 2008). In each case, inhibitor-contacting residues within a functionally essential, conformationally dynamic, secondary-structure element-BH-H N for Sal and 'switch 2' for Myx-are conserved from bacterial RNAP to human RNAP, but inhibitor-contacting residues in adjacent secondary-structure elements are not, allowing for selective inhibition of bacterial RNAP but not human RNAP.
Sal binds within a ∼2000 Å 3 pocket formed by the RNAP BH-H N , the RNAP fork loop, and the RNAP link region ( Figure 8B,C). Backbone atoms of residues that form the pocket have superimposible conformations in RNAP holoenzyme in the absence of Sal and in RNAP holoenzyme in complex with Sal, indicating that the pocket pre-exists in RNAP holoenzyme in the absence of Sal. The pocket opens at one end onto the RNAP secondary channel and the RNAP active-center 'i+1' nucleotide binding site ( Figure 8B,C). It seems likely that Sal enters the pocket from the RNAP secondary channel and/or the active-center i+1 nucleotide site.
Within the binding pocket, Sal residues 4, 5, 7, and 8 interact with the RNAP BH-H N , Sal residues 1-3 and 6-7 interact with the RNAP fork loop, and Sal residues 8 and 9 interact with the RNAP link region ( Figure 8B,C). Sal residue 9 is at the end of the pocket that opens onto the RNAP secondary channel and the active-center i+1 nucleotide binding site ( Figure 8B,C). The Sal residue-9 epoxide and methyl moieties extend into this opening and make no or limited interactions with RNAP ( Figure 8B,C).
The interactions observed in the structure suggest an opportunity for preparation of novel Sal analogs with improved potencies by semi-synthesis. The Sal residue-9 epoxide moiety is chemically reactive ( Figure 7A), can be altered without loss of activity ( Figure 7B), makes no or limited interactions with RNAP ( Figure 8B,C), and is directed toward the RNAP secondary channel and active-center i+1 nucleotide binding site ( Figure 8B,C). Accordingly, it should be possible to prepare novel Sal derivatives by semi-synthesis, introducing sidechains at the Sal residue-9 epoxide moiety that make additional interactions with RNAP, thereby potentially increasing RNAP-inhibitory activity and antibacterial activity (Figure 8-figure supplement 1). By way of example, sidechains that carry a negative charge would be positioned to make favorable electrostatic interactions with a cluster of positively-charged residues located in the RNAP secondary channel (the 'basic rim'; Vassylyev et al., 2007b;Zhang and Landick, 2009). By further way of example, a sidechain carrying a nucleotide, a nucleoside, or a nucleoside analog would be positioned to make highly favorable additional interactions with the RNAP active-center i+1 nucleotide binding site, potentially enabling highly potent RNAP-inhibitory activity and antibacterial activity.

Structural basis of transcription inhibition by Sal: Sal interacts with an 'open' (unbent) state of the bridge-helix N-terminal hinge and an 'open' (unfolded) state of the trigger loop
The crystal structure of the RNAP-Sal complex also defines effects of Sal on RNAP conformation (Figure 9). The crystal structure shows that Sal interacts with the RNAP BH-H N in an open (unbent) state ( Figure 9A), the same state that has been observed in previous crystal structures of RNAP and RNAP complexes (Zhang et al., 1999(Zhang et al., , 2012Campbell et al., 2001;Vassylyev et al., 2002Vassylyev et al., , 2007aVassylyev et al., , 2007bTemiakov et al., 2005;Tuske et al., 2005;Mukhopadhyay et al., 2008;Belogurov et al., 2009;Murakami, 2013;Zuo et al., 2013;Bae et al., 2013; Figure 9B). This conformation is different from the closed (bent) BH-H N conformation that has been observed in molecular dynamics simulations of nucleotide-addition reactions in transcription elongation complexes (Weinzierl, 2010;Kireeva et al., 2012;Nedialkov et al., 2013), and that has been postulated to serve as a critical intermediate in the bond-formation, pyrophosphate-release, and/or translocation reactions of the nucleotideaddition cycle (Hein and Landick, 2010;Weinzierl, 2010;Kireeva et al., 2012;Nedialkov et al., 2013). We conclude that Sal interacts with an open (unbent) BH-H N conformational state, and we propose that, through its interactions with that state, it stabilizes that state and prevents conformational dynamics required for nucleotide addition.
In the crystal structure of the RNAP-Sal complex, the RNAP trigger loop is disordered. Molecular modelling indicates that the structure of RNAP-Sal is compatible with the open (unfolded) trigger-loop conformations observed in crystal structures of RNAP and the transcription elongation complex without a bound NTP substrate (Zhang et al., 1999;Campbell et al., 2001;Vassylyev et al., 2002Vassylyev et al., , 2007aTemiakov et al., 2005;Tuske et al., 2005;Mukhopadhyay et al., 2008;Belogurov et al., 2009;Murakami, 2013;Zuo et al., 2013;Bae et al., 2013), but would be incompatible with the closed (folded) trigger loop conformation observed in the crystal structure of the transcription elongation complex with a bound NTP substrate (Vassylyev et al., 2007b; Figure 9C). We infer that Sal interacts with an open (unfolded) trigger-loop conformational state, and likely would prevent the formation of the closed (folded) trigger-loop conformational state. It is possible that effects of Sal on trigger-loop conformation may contribute to the mechanism of transcription inhibition by Sal. However, the results in Figure 5E show that the trigger loop is not essential for transcription inhibition by Sal, and therefore, although effects of Sal on trigger loop conformation may contribute to transcription inhibition by Sal, they cannot be essential for transcription inhibition by Sal.

Discussion
Bacterial RNAP is the functional cellular target of Sal The results in Figure 2 show that Sal inhibits RNAP in bacterial cells in culture, and that Sal-resistant mutations occur in RNAP subunit genes. The results establish that the RNAP is the functional cellular target of Sal, confirming the hypothesis that the RNAP-inhibitory activity of Sal is responsible for the antibacterial activity of Sal.

Sal interacts with the RNAP bridge-helix cap
The results in Figure 3 establish that transcription inhibition by Sal requires a determinant located within the RNAP active-center bridge-helix cap and comprising residues of the RNAP BH-H N , the RNAP F-loop, and the RNAP link region ('Sal target'). The results in Figure 4 establish that the Sal target is different from, and does not overlap, the targets of the previously characterized RNAP inhibitors Rif, Stl, CBR703, Myx, and Lpm. Consistent with the absence of overlap, mutants resistant to Sal are not cross-resistant with these other RNAP inhibitors, and, reciprocally, mutants resistant to these other RNAP inhibitors are not cross-resistant with Sal. Consistent with the absence of cross-resistance, co-administration of Sal and Rif, or of Sal and Myx, suppresses the emergence of spontaneous resistance, a finding that is significant since emergence of resistance limits the clinical application of Rif (Floss and Yu, 2005) and has been cited as a potential obstacle to the clinical development of Myx (Moy et al., 2011).

Sal inhibits nucleotide addition in transcription initiation and transcription elongation
The results in Figure 5 establish that Sal inhibits nucleotide addition in both transcription initiation and transcription elongation, interfering with one or more of the bond-formation, pyrophosphaterelease, or translocation sub-reactions of the nucleotide-addition cycle. The results in Figure 5 show that the mechanism of inhibition by Sal is different from the mechanisms of inhibition by the previously characterized RNAP inhibitors Rif, Stl, Myx, and Lpm; and further results in Figure 4B suggest, although do not prove, that the mechanism of Sal also is different from the mechanism of the previously characterized RNAP inhibitor CBR703.

Sal allosterically inhibits nucleotide addition through interaction with the bridge-helix cap trapping an 'open' (unbent) state of the bridge-helix N-terminal hinge
The crystal structures of RNAP-Sal and RNAP-Sal-Br complexes in Figures 6-8 confirm that Sal binds within the RNAP bridge-helix cap, making interactions with residues of the BH-H N , the F-loop, and the link region. The structures establish that Sal does not contact, or clash with, the RNAP activecenter catalytic Mg 2+ ion or the RNAP residues that interact with the DNA template, the RNA product, or the NTP substrate, indicating that Sal interferes with nucleotide addition allosterically. The structures further reveal that Sal interacts with an open (unbent) state of the BH-H N ( Figure 8A). We propose that Sal allosterically inhibits nucleotide addition by interacting with and stabilizing the open (unbent) state of the BH-H N .

Sal as a chemical probe of bridge-helix N-terminal hinge conformation
Sal is the first RNAP inhibitor that has been proposed to function through effects on conformational dynamics of the BH-H N . We suggest that Sal will find use as a research tool for dissection of mechanistic and structural aspects of BH-H N conformational dynamics.

Sal as a starting point for antibacterial drug discovery
The semi-synthesis of Sal-Br from SalA ( Figure 7A) shows that the SalA epoxide moiety provides a chemical reactivity that can be exploited for semisynthesis of novel Sal analogs. The retention of RNAP inhibitory activity and antibacterial activity by Sal-Br ( Figure 7B) shows that semi-synthetic modifications at the SalA epoxide moiety can be tolerated without loss of potency. The crystal structures of RNAP-Sal and RNAP-Sal-Br (Figures 6-8) show that the SalA epoxide moiety makes no or limited interactions with RNAP and is located at the entrance to the Sal-binding pocket, directed towards the RNAP secondary channel and RNAP active-center i+1 nucleotide binding site ( Figure 8B,C; Figure 8-figure supplement 1). These findings, together with the published total synthesis of SalA (Tan and Ma, 2008), set the stage for rational, structure-based design of novel semi-synthetic and fully synthetic Sal analogs with increased potency. Introduction at the SalA epoxide moiety of a sidechain with negatively-charged functionality should enable new, energetically favorable, electrostatic interactions with positively-charged 'basic-rim' residues in the RNAP secondary channel. Introduction at the Sal epoxide moiety of a nucleotide or nucleoside analog, should enable new, energetically favorable, interactions with the RNAP active-center i+1 nucleotide binding site. Covalently linking Sal to a nucleotide or nucleoside analog is expected to yield a bipartite inhibitor that interacts simultaneously with the Sal binding pocket and the active-center i+1 nucleotide binding site, and therefore, that potentially exhibits a very high affinity of binding and a very high potency of inhibition. Reciprocally, equipping a nucleoside-analog RNAP inhibitor with chemical functionality able to interact with the Sal pocket should provide a means both to increase potency of the nucleoside-analog inhibitor and to introduce selectivity for inhibition of bacterial RNAP vs inhibition of human RNAP.

Materials and methods Sal
SalA and SalB were prepared from cultures of Streptomyces sp. CNB-091 as in Moore et al. (1999).
MICs for mammalian cells (Vero E6) in culture were quantified using CellTiter96 assay (Promega, Madison, WI; procedures as specified by the manufacturer).

Spontaneous Sal-resistant mutants
E. coli D21f2tolC was cultured to saturation in 5 ml LB broth at 37°C, cultures were centrifuged, and cell pellets (∼2 × 10 9 cells) were re-suspended in 50 μl LB broth and plated on LB agar (Sambrook and Russell, 2001) containing 0.6 or 1.2 μg/ml SalA (2 × MIC or 4 × MIC under these conditions), and incubated 24-48 hr at 37°C. Sal-resistant mutants were identified by the ability to form colonies on this medium and were confirmed by re-streaking on the same medium.

Induced Sal-resistant mutants
Induced Sal-resistant mutants were isolated using procedures analogous to those used for isolation of induced Myx-resistant mutants in Mukhopadhyay et al. (2008). Random mutagenesis of rpoB plasmid pRL706 (Severinov et al., 1997) and rpoC plasmid pRL663 (Wang et al., 1995) was performed by use of PCR amplification, exploiting the baseline error rate of PCR amplification. Mutagenesis reactions were performed using the QuikChange Site-Directed Mutagenesis Kit (Agilent/Stratagene), with pRL706 as template and oligodeoxyribonucleotide forward and reverse primers corresponding to nucleotides 427-446 of lacI (5′-GTTCCGGCGTTATTTCTTGA-3′ and 5′-TCAAGAAATAACGCCGGAAC-3′), or with pRL663 as template and oligodeoxyribonucleotide forward and reverse primers corresponding to nucleotides 217-246 of lacI (5′-CTGCACGCGCCGTCGAAAATTGTCGCGGCG-3′ and 5′-CGCCGCGAC AATTTTCGACGGCGCGTGCAG-3′) (primers at 160 nM; all other components at concentrations as specified by the manufacturer). Mutagenized plasmid DNA was introduced by transformation into E. coli XL1-Blue (Agilent/Stratagene). Transformants (∼5 × 10 3 cells) were applied to LB-agar plates containing 200 μg/ml ampicillin, plates were incubated 16 hr at 37°C, and plasmid DNA was prepared from the pooled resulting colonies. The resulting passaged random-mutagenesis library was pooled in a 1/1 (wt/wt) ratio with pooled passaged saturation-mutagenesis libraries of Mukhopadhyay et al. (2004), Tuske et al. (2005), andMukhopadhyay et al. (2008), and the resulting pooled mutagenized plasmid DNA was introduced by transformation into E. coli D21f2tolC. Transformants (∼10 3 cells) were applied to LB-agar plates containing 0.4 μg/ml SalA (for pRL706) or 1 μg/ml SalA (for pRL663), 200 μg/ml ampicillin, and 1 mM IPTG, and plates were incubated 24-48 hr at 37°C. Sal-resistant mutants were identified by the ability to form colonies on this medium, were confirmed by re-streaking on the same medium, and were demonstrated to contain plasmid-linked Sal-resistant mutations by preparing plasmid DNA, transforming E. coli D21f2tolC with plasmid DNA, and plating transformants on the same medium. Nucleotide sequences of rpoB and rpoC were determined by Sanger sequencing (eight primers per gene).

Resistance levels
Resistance levels of Sal-resistant mutants were quantified by performing broth microdilution assays. Single colonies were inoculated into 5 ml LB broth (LB broth containing 200 μg/ml ampicillin for induced mutants and wild-type controls for induced mutants) and incubated at 37°C with shaking until OD 600 = 0.4-0.8. (At this point, IPTG was added to a final concentration of 1 mM for induced mutants and wild-type controls for induced mutants, and the cultures were grown for an additional 1 hr at 37°C with shaking.) Diluted aliquots (∼5 × 10 4 cells in 97 μl LB broth; LB broth containing 200 μg/ml ampicillin and 1 mM IPTG for induced mutants and wild-type controls for induced mutants) were dispensed into wells of a 96-well plate, were supplemented with 3 μl of a twofold dilution series of SalA or SalB in methanol (final concentrations = 0.0015-50 μg/ml), or 3 μl of a solvent blank, and were incubated 16 hr at 37°C with shaking. The MIC was defined as the lowest tested concentration of SalA that inhibited bacterial growth by ≥90%.
Cross-resistance levels of Rif-resistant mutants, Myx-resistant mutants, and Lpm-resistant mutants (mutations transferred from pRL706 or pRL663 derivatives [Ebright, 2005;Mukhopadhyay et al., 2008;DD, S Ismail and RHE, unpublished] to the chromosome of E. coli D21f2tolC by λ-Red-mediated recombineering [procedures essentially as in Datsenko and Wanner, 2000, but using transformation rather than electroporation]) were determined analogously to resistance levels of spontaneous Salresistant mutants. Cross-resistance levels of Stl-resistant mutants and CBR703-resistant mutants (mutations on pRL706 and pRL663 derivatives; Tuske et al., 2005;X Wang and RHE, unpublished) were determined analogously to resistance levels of induced Sal-resistant mutants.

Structure determination: data collection and reduction
Diffraction data were collected from cryo-cooled crystals at Cornell High Energy Synchrotron Source beamline F1 and at Brookhaven National Laboratory beamline X25. Data were processed using HKL2000 (Otwinowski and Minor, 1997).

Structure determination: structure solution and refinement
The structure of E. coli RNAP holoenzyme was solved by molecular replacement using AutoMR (McCoy et al., 2007;Adams et al., 2010). The search model was generated by starting with the crystal structure of T. thermophilus RNAP-promoter open complex (PDB 4G7H; Zhang et al., 2012), deleting DNA and non-conserved protein domains, modelling E. coli α I and α II subunit N-terminal domains by superimposing the crystal structure of E. coli α N-terminal domain dimer (PDB 1BDF; Zhang and Darst, 1998), and modelling E. coli β, β', ω, and σ 70 subunits using Sculptor (Bunkóczi and Read, 2011; backbone and sidechain atoms for identical residues; backbone and Cβ atoms for non-identical residues). Two RNAP molecules are present in the asymmetric unit. Crystal structures of E. coli α subunit C-terminal domain (PDB 3K4G;Lara-González et al., 2010), the E. coli β subunit β2-βi4 and βflap-βi9 domains (PDB 3LTI and PDB 3LU0; Opalka et al., 2010), and E. coli σ 70 region 2 (PDB 1SIG; Malhotra et al., 1996) were fitted manually to the (Fo-Fc) difference electron density map. Early-stage refinement of the structure was performed using Phenix (Adams et al., 2010) and included rigid-body refinement of each RNAP molecule in the asymmetric unit, followed by rigid-body refinement of each subunit of each RNAP molecule, followed by rigid-body refinement of 216 segments of each RNAP molecule, followed by group B-factor refinement with one B-factor group per residue, using Phenix (methods as in Zhang et al., 2012). Density modification, including non-crystallographicsymmetry averaging and solvent flattening using a locally modified version of DM (Collaborative Computational Project, 1994), was performed to remove model bias and to improve phases. The resulting maps allowed segments that were not present in the search model to be built manually using Coot (Emsley et al., 2010). Cycles of iterative model building with Coot and refinement with Phenix improved the model. The final E. coli RNAP holoenzyme model, refined to Rwork and Rfree of 0.276 and 0.325, respectively, has been deposited in the PDB with accession code 4MEY (Supplementary file 2).
The structures of the E. coli RNAP-SalA and RNAP-Sal-Br complexes were solved by molecular replacement in AutoMR, using the above crystal structure of E. coli RNAP holoenzyme as the search model. For each structure, after rigid-body refinement with 216 domains, an electron density feature corresponding to one molecule of SalA per holoenzyme was clearly visible in the (Fo-Fc) difference map. A structural model of SalA derived from the crystal structure of SalB (CSD 50962;Trischman et al., 1994; enantiomorph corrected based on Moore et al., 1999) was fitted to the (Fo-Fc) difference map with minor adjustments of SalA conformation, and the fit was confirmed by the position of the peak of Br anomalous difference density for the RNAP-Sal-Br complex. The final E. coli RNAP-SalA complex model, refined to Rwork and Rfree of 0.286 and 0.325, respectively, has been deposited in the PDB with accession code 4MEX (Supplementary file 2).