Pseudouridimycin (PUM) is a C-nucleoside/peptide antibiotic that selectively inhibits bacterial RNA polymerase (RNAP) and exhibits potent activity against drug-resistant pathogens. However, PUM suffers from chemical instability due to self-immolative cleavage of its central hydroxamate bond. Here, we employed cryo-electron microscopy to determine structures of PUM (1) and a chemically stabilized des-hydroxy analog of PUM (2a) bound to an Escherichia coli RNAP transcription complex. Guided by the observed bound conformation, we developed an efficient solid-phase synthesis of 50 des-hydroxy PUM analogs modified at the Gln residue and Gdn-Gly tail. Several analogs retained low-micromolar RNAP-inhibitory activity, with a para-substituted phenyl amidine analog (54) emerging as the most potent inhibitor (IC50 = 0.95 μM). These results establish a versatile synthetic platform and structural framework for optimizing stabilized PUM derivatives and provide a foundation for the development of RNAP-targeted therapeutics against resistant bacterial pathogens.

Structures recently have been reported of molecular assemblies that mediate transcription-translation coupling in Escherichia coli. In these molecular assemblies, termed "coupled transcription-translation complexes" or "TTC-B," RNA polymerase (RNAP) directly interacts with the ribosome, the transcription elongation factor NusG or its paralog RfaH forms a bridge between RNAP and ribosome, and the transcription elongation factor NusA optionally forms a second bridge between RNAP and ribosome. Here, we report structures of coupled transcription-translation complexes having mRNA spacers between RNAP and ribosome longer than the maximum-length mRNA spacer compatible with formation of TTC-B. The results define a class of coupled transcription-translation complex, termed "TTC-LC," where "LC" denotes "long-range coupling." TTC-LC differs from TTC-B by a ~60° rotation and ~70 Å translation of RNAP relative to ribosome, resulting in loss of direct interactions between RNAP and ribosome and creation of a ~70 Å gap between RNAP and ribosome. TTC-LC accommodates long mRNA spacers by looping out mRNA from the gap between RNAP and ribosome. We present evidence that TTC-LC is a functional intermediate in assembling and disassembling TTC-B, mediating pre-TTC-B transcription-translation coupling before a ribosome catches up to RNAP, and mediating post-TTC-B transcription-translation coupling after a ribosome stops moving and RNAP continues moving. We show that TTC-B, but not TTC-LC, is severely defective in RNA-hairpin-dependent transcription termination, and that both TTC-B and TTC-LC are severely defective in Rho-dependent transcription termination.

S ingle-molecule p icometer- r esolution n anopore tweezers (SPRNT) enables monitoring of translocation of a nucleic-acid motor protein on a nucleic-acid track with sequence registration, sub-nucleotide spatial resolution, sub-millisecond temporal resolution, and the ability to apply forces that assist or oppose translocation. Recently, we used SPRNT to analyze the translocation of single molecules of Escherichia coli RNA polymerase relative to the DNA template strand during transcription elongation, and we directly detected sequence-dependent pausing and formation of a "half-translocated state" at the E. coli yrbL consensus pause element. Here, we apply SPRNT to analyze the translocation of single molecules of yeast RNA polymerase II (Pol II) relative to the DNA template strand during transcription elongation with single-nucleotide spatial resolution and millisecond-scale temporal resolution at biologically relevant, saturating substrate concentrations; we compare translocation by intact, 12-subunit Pol II to translocation by a 10-subunit Pol II sub-assembly lacking the dissociable Rpb4-Rpb7 Pol II "stalk"; and we assess possible pausing by intact Pol II and stalk-less Pol II at the E. coli yrbL consensus pause element. The results show that intact Pol II elongates more rapidly than stalk-less Pol II and show that neither intact Pol II nor stalk-less Pol II pauses at the E. coli yrbL consensus pause element. One-sentence summary:Nanopore tweezers enable monitoring of translocation of RNA polymerase II relative to DNA in transcription elongation with single-nucleotide spatial resolution and millisecond-scale temporal resolution at biologically relevant, saturating substrate concentrations.

The Mycobacterium tuberculosis RNA polymerase (MtbRNAP) is the target of the first-line anti-tuberculosis inhibitor rifampin; however, the emergence of rifampin resistance necessitates the development of new antibiotics. Here, we communicate the first single-molecule characterization of MtbRNAP elongation and its inhibition by three diverse small-molecule inhibitors: N(α)-aroyl-N-aryl-phenylalaninamide (D-IX216), streptolydigin (Stl), and pseudouridimycin (PUM) using high-resolution optical tweezers. Compared to Escherichia coli RNA polymerase (EcoRNAP), MtbRNAP transcribes more slowly, has similar mechanical robustness, and only weakly recognizes E. coli pause sequences. The three small-molecule inhibitors of MtbRNAP exhibit strikingly different effects on transcription elongation. In the presence of D-IX216, which inhibits RNAP active-center bridge-helix motions required for nucleotide addition, the enzyme exhibits transitions between slowly and super-slowly elongating inhibited states. Stl, which inhibits the RNAP trigger-loop motions also required for nucleotide addition, inhibits RNAP primarily by inducing pausing and backtracking. PUM, a nucleoside analog of UTP, in addition to acting as a competitive inhibitor, induces the formation of slowly elongating RNAP-inhibited states. Our results indicate that the three classes of small-molecule inhibitors affect the enzyme in distinct ways and show that the combination of Stl and D-IX216, which both target the RNAP bridge helix, has a strong synergistic effect on the enzyme.

A "σ cycle" in which the initiation factor σ associates with RNA polymerase (RNAP) core enzyme to permit transcription initiation and dissociates from RNAP core enzyme to permit transcription elongation, has been proposed to occur and to be an essential step for σ-exchange, with all principal σ factors from all bacteria. These proposals were based on studies of the principal σ factor of Escherichia coli, σ70, which generally, albeit not obligatorily, is released from RNAP upon the transition from transcription initiation to elongation. Here, we show that, in contrast to E. coli σ70, the Bacillus subtilis principal σ factor, σA, is not released and is retained on RNAP core throughout transcription elongation. We further show that a mutant E. coli σ70 derivative lacking σ region 1.1 (σ R1.1) is not released and is retained on RNAP core throughout transcription elongation. We also observe that B. subtilis σA and the mutant E. coli σ70 derivative lacking σ R1.1 interact much more stably with RNAP than full-length E. coli σ70. Our results indicate that the σ cycle is not a universal phenomenon in bacteria.

Single-molecule picometer-resolution nanopore tweezers (SPRNT) enables monitoring of translocation of a nucleic-acid motor protein on a nucleic-acid track with sequence registration, sub-nucleotide spatial resolution, sub-millisecond temporal resolution, and the ability to apply assisting forces. Previous work has demonstrated the ability of SPRNT to monitor the translocation of Escherichia coli RNA polymerase (RNAP) relative to the DNA template strand in transcription elongation and has directly detected a half-translocated state in sequence-dependent transcriptional pausing (Nova, I., Craig, J., Mazumder, A., Laszlo, A., Derrington, I., Noakes, M., Brinkerhoff, H., Yang, S., Vahedian-Movahed, H., Li, L., Zhang, Y., Bowman, J., Mount, J., Huang, J., Ebright., R., and Gundlach, J., in preparation).

Rho is a ring-shaped hexameric ATP-dependent molecular motor. Together with the transcription elongation factor NusG, Rho mediates factor-dependent transcription termination and transcription-translation-coupling quality control in Escherichia coli(1-4). Here we report the preparation of complexes that are functional in factor-dependent transcription termination from Rho, NusG, RNA polymerase (RNAP), and synthetic nucleic acid scaffolds, and we report cryogenic electron microscopy structures of the complexes. The structures show that functional factor-dependent pre-termination complexes contain a closed-ring Rho hexamer; have RNA threaded through the central channel of Rho; have 60 nucleotides of RNA interacting sequence-specifically with the exterior of Rho and 6 nucleotides of RNA interacting sequence-specifically with the central channel of Rho; have Rho oriented relative to RNAP such that ATP-dependent translocation by Rho exerts mechanical force on RNAP; and have NusG bridging Rho and RNAP. The results explain five decades of research on Rho and provide a foundation for understanding Rho's function.

The NusG paralog RfaH mediates bacterial transcription-translation coupling on genes that contain a DNA sequence element, termed an ops site, required for pausing RNA polymerase (RNAP) and for loading RfaH onto the paused RNAP. Here we report cryo-EM structures of transcription-translation complexes (TTCs) containing RfaH. The results show that RfaH bridges RNAP and the ribosome, with the RfaH N-terminal domain interacting with RNAP, and with the RfaH C-terminal domain interacting with the ribosome. The results show that the distribution of translational and orientational positions of RNAP relative to the ribosome in RfaH-coupled TTCs is more restricted than in NusG-coupled TTCs, due to the more restricted flexibility of the RfaH interdomain linker. The results further show that the structural organization of RfaH-coupled TTCs in the "loading state," in which RNAP and RfaH are located at the ops site during formation of the TTC, is the same as the structural organization of RfaH-coupled TTCs in the "loaded state," in which RNAP and RfaH are located at positions downstream of the ops site during function of the TTC. The results define the structural organization of RfaH-containing TTCs and set the stage for analysis of functions of RfaH during translation initiation and transcription-translation coupling. One sentence summary:Cryo-EM reveals the structural basis of transcription-translation coupling by RfaH.

Pseudouridimycin (PUM) is a microbially produced C-nucleoside dipeptide that selectively targets the nucleotide addition site of bacterial RNA polymerase (RNAP) and that has a lower rate of spontaneous resistance emergence relative to current drugs that target RNAP. Despite its promising biological profile, PUM undergoes relatively rapid decomposition in buffered aqueous solutions. Here, we describe the synthesis, RNAP-inhibitory activity, and antibacterial activity of chemically stabilized analogues of PUM. These analogues feature targeted modifications that mitigate guanidine-mediated hydroxamate bond scission. A subset of analogues in which the central hydroxamate is replaced with amide or hydrazide isosteres retain the antibacterial activity of the natural product.

We report the synthesis of the antimicrobial cyclodepsipeptides marformycin A (1) and marformycin D (2) using a solid-phase approach. A scalable solution-phase synthesis of the γ-hydroxypiperazic acid subunit in 2, starting from cis-hydroxyproline, is also described. Structural analysis of 1 and its Leu-epi congener demonstrates conformational differences that may underlie their divergent antimicrobial activities. The described approach enables further development of conformation-activity relationships within this class of depsipeptide natural products.

RNA polymerases (RNAPs) carry out the first step in the central dogma of molecular biology by transcribing DNA into RNA. Despite their importance, much about how RNAPs work remains unclear, in part because the small (3.4 Angstrom) and fast (~40 ms/nt) steps during transcription were difficult to resolve. Here, we used high-resolution nanopore tweezers to observe the motion of single Escherichia coli RNAP molecules as it transcribes DNA ~1,000 times improved temporal resolution, resolving single-nucleotide and fractional-nucleotide steps of individual RNAPs at saturating nucleoside triphosphate concentrations. We analyzed RNAP during processive transcription elongation and sequence-dependent pausing at the yrbL elemental pause sequence. Each time RNAP encounters the yrbL elemental pause sequence, it rapidly interconverts between five translocational states, residing predominantly in a half-translocated state. The kinetics and force-dependence of this half-translocated state indicate it is a functional intermediate between pre- and post-translocated states. Using structural and kinetics data, we show that, in the half-translocated and post-translocated states, sequence-specific protein–DNA interaction occurs between RNAP and a guanine base at the downstream end of the transcription bubble (core recognition element). Kinetic data show that this interaction stabilizes the half-translocated and post-translocated states relative to the pre-translocated state. We develop a kinetic model for RNAP at the yrbL pause and discuss this in the context of key structural features.

Bacterial transcription and translation are frequently linked through a transcribing RNA polymerase following the leading ribosome, a process termed transcription-translation coupling (TTC). Two distinct TTC structures, the collied-TTC (TTC-A) and the coupled-TTC (TTC-B), have been reported, but the biological significance of both complexes is still subject to uncertainty. Furthermore, the ribosomes in all of them are in a static state. The underlying translation dynamics within these TTCs remain elusive. Here we report cryo-EM structures of transcription-translation complexes from E.coli at various stages of the translation elongation cycle. The results show five states of TTC-B representing the whole stages of translation elongation cycle. TTC-B is compatible with ribosome conformational changes during the translation elongation and coordinates transcription elongation with translation elongation. The results show two distinct TTC-A in translational pre-translocation and translocation intermediate states in which the RNAP becomes unstable as the translation progresses. The results further show that TTC-A is the key state where the ribsomes could exert mechanical force on RNAP leading to the potential transition between TTC-A and TTC-B within mRNA spacer ranging from 7 to 9 codons and the TTC-A-dependent transcription termination within mRNA spacer shorter than 7 codons. Taken together, our results provide a comprehensive blueprint detailing the dynamic interplay of translation coupled with transcription.### Competing Interest StatementThe authors have declared no competing interest.

Chlamydiae are obligate intracellular bacteria that replicate only inside eukaryotic cells. Previously, it has not been possible to identify a candidate gene encoding the chlamydial RNA polymerase ω subunit, and it has been hypothesized that the chlamydial RNA polymerase ω subunit was lost in the evolutionary process through which Chlamydiae reduced their genome size and proteome sizes to adapt to an obligate intracellular lifestyle.

TNP-2198, a stable conjugate of a rifamycin pharmacophore and a nitroimidazole pharmacophore, has been designed, synthesized, and evaluated as a novel dual-targeted antibacterial agent for the treatment of microaerophilic and anaerobic bacterial infections. TNP-2198 exhibits greater activity than a 1:1 molar mixture of the parent drugs and exhibits activity against strains resistant to both rifamycins and nitroimidazoles. A crystal structure of TNP-2198 bound to a Mycobacterium tuberculosis RNA polymerase transcription initiation complex reveals that the rifamycin portion of TNP-2198 binds to the rifamycin binding site on RNAP and the nitroimidazole portion of TNP-2198 interacts directly with the DNA template-strand in the RNAP active-center cleft, forming a hydrogen bond with a base of the DNA template strand. TNP-2198 is currently in Phase 2 clinical development for the treatment of Helicobacter pylori infection, Clostridioides difficile infection, and bacterial vaginosis.

In transcription initiation, RNA polymerase (RNAP) binds to promoter DNA, unwinds a turn of promoter DNA to yield an RNAP–promoter open complex containing an unwound “transcription bubble,” and selects a transcription start site (TSS). In the next step of initiation, termed “initial transcription,” RNAP remains bound to the promoter and synthesizes an RNA product of a threshold length of approximately 11–15 nucleotides. In the final step of initiation, termed “promoter escape,” RNAP breaks free of the promoter to yield a transcription elongation complex that synthesizes the rest of the RNA product. As a result of research over the last two decades, we now have a detailed mechanistic understanding of TSS selection, and we now understand broad outlines of initial transcription and promoter escape. Here we review the current understanding of TSS selection, initial transcription, and promoter escape, focusing on these processes as they occur in the best characterized example, transcription initiation by Escherichia coli RNAP-σ70 holoenzyme, but also summarizing these processes as they occur in eukaryotic RNAP I, II, and III.

ABSTRACT Gene transcription in bacteria is carried out by the multisubunit RNA polymerase (RNAP), which is composed of a catalytic core enzyme and a promoter-recognizing σ factor. RNAP core enzyme comprises two α subunits, one β subunit, one β’ s subunit, and one ω (omega) subunit. Across multiple bacterial taxa, the RNAP ω subunit plays critical roles in the assembly of RNAP core enzyme and in other cellular functions, including regulation of bacterial growth, stress response, and biofilm formation. However, for several intracellular bacterium, including the obligate intracellular bacterium Chlamydia , no RNAP ω subunit previously has been identified. Here, we report the identification of Chlamydia trachomatis hypothetical protein CTL0286 as the chlamydial RNAP ω ortholog, based on sequence, synteny, and AlphaFold and AlphaFold-Multimer three-dimensional-structure predictions. We conclude that CTL0286 functions as the previously missing chlamydial ω ortholog. Extensions of our analysis indicate that all obligate intracellular bacteria have ω orthologs. IMPORTANCE Chlamydiae are common mammalian pathogens. Chlamydiae have a unique developmental cycle characterized with an infectious but nondividing elementary body (EB), which can temporarily survive outside host cells, and a noninfectious reticulate body (RB), which replicates only intracellularly. Chlamydial development inside host cells can be arrested during persistence in response to adverse environmental conditions. Transcription plays a central role in the progression of the chlamydial developmental cycle as well as entry into and recovery from persistence. The identification of the elusive ω subunit of chlamydial RNAP makes possible future study of its regulatory roles in gene expression during chlamydial growth, development, and stress responses. This discovery also paves the way to prepare and study the intact chlamydial RNAP and its interactions with inhibitors in vitro .

Lambdoid bacteriophage Q proteins are transcription antipausing and antitermination factors that enable RNA polymerase (RNAP) to read through pause and termination sites. Q proteins load onto RNAP engaged in promoter-proximal pausing at a Q binding element (QBE) and adjacent sigma-dependent pause element to yield a Q-loading complex, and they translocate with RNAP as a pausing-deficient, termination-deficient Q-loaded complex. In previous work, we showed that the Q protein of bacteriophage 21 (Q21) functions by forming a nozzle that narrows and extends the RNAP RNA-exit channel, preventing formation of pause and termination RNA hairpins. Here, we report atomic structures of four states on the pathway of antitermination by the Q protein of bacteriophage λ (Qλ), a Q protein that shows no sequence similarity to Q21 and that, unlike Q21, requires the transcription elongation factor NusA for efficient antipausing and antitermination. We report structures of Qλ, the Qλ-QBE complex, the NusA-free pre-engaged Qλ-loading complex, and the NusA-containing engaged Qλ-loading complex. The results show that Qλ, like Q21, forms a nozzle that narrows and extends the RNAP RNA-exit channel, preventing formation of RNA hairpins. However, the results show that Qλ has no three-dimensional structural similarity to Q21, employs a different mechanism of QBE recognition than Q21, and employs a more complex process for loading onto RNAP than Q21, involving recruitment of Qλ to form a pre-engaged loading complex, followed by NusA-facilitated refolding of Qλ to form an engaged loading complex. The results establish that Qλ and Q21 are not structural homologs and are solely functional analogs.

Rifamycin antibiotics are a valuable class of antimicrobials for treating infections by mycobacteria and other persistent bacteria owing to their potent bactericidal activity against replicating and non-replicating pathogens. However, the clinical utility of rifamycins against Mycobacterium abscessus is seriously compromised by a novel resistance mechanism, namely, rifamycin inactivation by ADP-ribosylation. Using a structure-based approach, we rationally redesign rifamycins through strategic modification of the ansa-chain to block ADP-ribosylation while preserving on-target activity. Validated by a combination of biochemical, structural, and microbiological studies, the most potent analogs overcome ADP-ribosylation, restored their intrinsic low nanomolar activity and demonstrated significant in vivo antibacterial efficacy. Further optimization by tuning drug disposition properties afforded a preclinical candidate with remarkable potency and an outstanding pharmacokinetic profile.

AbstractIn σ-dependent transcriptional pausing, the transcription initiation factor σ, translocating with RNA polymerase (RNAP), makes sequence-specific protein-DNA interactions with a promoter-like sequence element in the transcribed region, inducing pausing. It has been proposed that, in σ-dependent pausing, the RNAP active center can access off-pathway “backtracked” states that are substrates for the transcript-cleavage factors of the Gre family, and on-pathway “scrunched” states that mediate pause escape. Here, using site-specific protein-DNA photocrosslinking to define positions of the RNAP trailing and leading edges and of σ relative to DNA at the λPR’ promoter, we show directly that σ-dependent pausing in the absence of GreB in vitro predominantly involves a state backtracked by 2-4 bp, and that σ-dependent pausing in the presence of GreB in vitro and in vivo predominantly involves a state scrunched by 2-3 bp. Analogous experiments with a library of 47 (∼16,000) transcribed-region sequences show that the state scrunched by 2-3 bp--and only that state--is associated with the consensus sequence, T-3N-2Y-1G+1, (where -1 corresponds to the position of the RNA 3’ end), which is identical to the consensus for pausing in initial transcription, and which is related to the consensus for pausing in transcription elongation. Experiments with heteroduplex templates show that sequence information at position T-3 resides in the DNA nontemplate strand. A cryo-EM structure of a complex engaged in σ-dependent pausing reveals positions of DNA scrunching on the DNA nontemplate and template strands and suggests that position T-3 of the consensus sequence exerts its effects by facilitating scrunching.