Structural studies of eukaryotic RNA polymerase II (Pol II) transcription often rely on in vitro assembly, which may not fully represent native conditions. To investigate Pol II transcription in metazoan cells, we developed a method to isolate native transcription complexes from Drosophila melanogaster embryos using FLAG-tag affinity purification and Micrococcal Nuclease treatment. Cryo-EM and proteomics studies revealed diverse transcription complexes and nucleosomes, including a metazoan Rpb4/Rpb7 stalk-less Pol II elongation complex and a hexameric nucleosome lacking an H2A/H2B dimer. Notably, nucleosome is found only downstream of the nucleosome elongation complex, underscoring it as a major energy barrier and a time-consuming step during Pol II progression through chromatin. Proteomics identified co-purified factors involved in transcription initiation, elongation, and RNA modification. This study provides a framework for investigations of transcription in cells, paving the way for future studies of transient and minor complexes.

DNA polymerase δ (Pol δ) is a key enzyme in eukaryotic DNA replication and genome maintenance, essential for lagging strand synthesis, leading strand initiation, and DNA repair. While human Pol δ exhibits high activity and processivity in its holoenzyme form complexed with proliferating cell nuclear antigen (PCNA), it shows minimal DNA synthesis activity without PCNA, the molecular basis of which remains unclear. Here, we present the cryo-EM structure of the apo-form human Pol δ, comprising the catalytic subunit p125 and regulatory subunits p66, p50, and p12, at an overall resolution of 3.65 Å. We identified an acidic α-helix at the N terminus of p125, which occupies the single-stranded DNA-binding cavity within the polymerase domain in the apo-form Pol δ. This interaction likely inhibits DNA binding in the absence of PCNA, explaining the low activity of apo-form Pol δ. The acidic α-helix is absent in yeast Pol δ, providing a molecular explanation for species-specific differences in PCNA-independent Pol δ activity. These findings provide critical insights into the regulatory mechanisms of Pol δ and its reliance on PCNA for efficient DNA synthesis.

The processing of the Coronavirus polyproteins pp1a and pp1ab by the main protease Mpro to produce mature proteins is a crucial event in virus replication and a promising target for antiviral drug development. Mpro cleaves polyproteins in a defined order, but how Mpro and/or the polyproteins determine the order of cleavage remains enigmatic due to a lack of structural information about polyprotein-bound Mpro. Here, we present the cryo-EM structures of SARS-CoV-2 Mpro in an apo form and in complex with the nsp7-10 region of the pp1a polyprotein. The complex structure shows that Mpro interacts with only the recognition site residues between nsp9 and nsp10, without any association with the rest of the polyprotein. Comparison between the apo form and polyprotein-bound structures of Mpro highlights the flexible nature of the active site region of Mpro, which allows it to accommodate ten recognition sites found in the polyprotein. These observations suggest that the role of Mpro in selecting a preferred cleavage site is limited and underscores the roles of the structure, conformation, and/or dynamics of the polyproteins in determining the sequence of polyprotein cleavage by Mpro. The research was supported in part by the National Cancer Institute's National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research under contract HSSN261200800001E. This work was supported by an NIH grant (R35 GM131860) to K. S. M.

Cyanobacteria and evolutionarily related chloroplasts of algae and plants possess unique RNA polymerases (RNAPs) with characteristics that distinguish from canonical bacterial RNAPs. The largest subunit of cyanobacterial RNAP (cyRNAP) is divided into two polypeptides, β'1 and β'2, and contains the largest known lineage-specific insertion domain, Si3, located in the middle of the trigger loop and spans approximately half of the β'2 subunit. In this study, we present the X-ray crystal structure of Si3 and the cryo-EM structures of the cyRNAP transcription elongation complex plus the NusG factor with and without incoming nucleoside triphosphate (iNTP) bound at the active site. Si3 has a well-ordered and elongated shape that exceeds the length of the main body of cyRNAP, fits into cavities of cyRNAP and shields the binding site of secondary channel-binding proteins such as Gre and DksA. A small transition from the trigger loop to the trigger helix upon iNTP binding at the active site results in a large swing motion of Si3; however, this transition does not affect the catalytic activity of cyRNAP due to its minimal contact with cyRNAP, NusG or DNA. This study provides a structural framework for understanding the evolutionary significance of these features unique to cyRNAP and chloroplast RNAP and may provide insights into the molecular mechanism of transcription in specific environment of photosynthetic organisms.

The processing of the Coronavirus polyproteins pp1a and pp1ab by the main protease Mpro to produce mature proteins is a crucial event in virus replication and a promising target for antiviral drug development. Mpro cleaves polyproteins in a defined order, but how Mpro and/or the polyproteins determine the order of cleavage remains enigmatic due to a lack of structural information about polyprotein-bound Mpro. Here, we present the cryo-EM structures of SARS-CoV-2 Mpro in an apo form and in complex with the nsp7-10 region of the pp1a polyprotein. The complex structure shows that Mpro interacts with only the recognition site residues between nsp9 and nsp10, without any association with the rest of the polyprotein. Comparison between the apo form and polyprotein-bound structures of Mpro highlights the flexible nature of the active site region of Mpro, which allows it to accommodate ten recognition sites found in the polyprotein. These observations suggest that the role of Mpro in selecting a preferred cleavage site is limited and underscores the roles of the structure, conformation, and/or dynamics of the polyproteins in determining the sequence of polyprotein cleavage by Mpro.

Hydrothermal systems form at divergent and convergent boundaries of lithospheric plates and within plates due to weakened crust and mantle plumes, playing host to diverse microbial ecosystems. Little is known of how differences in tectonic setting influence the geochemical and microbial compositions of these hydrothermal ecosystems. Here, coordinated geochemical and microbial community analyses were conducted on 87 high-temperature (>65°C) water and sediment samples from hot springs in Yellowstone National Park, Wyoming, USA (n = 41; mantle plume setting), Iceland (n = 41, divergent boundary), and Japan (n = 5; convergent boundary). Region-specific variation in geochemistry and sediment-associated 16S rRNA gene amplicon sequence variant (ASV) composition was observed, with 16S rRNA gene assemblages being nearly completely distinguished by region and pH being the most explanatory parameter within regions. Several low abundance ASVs exhibited cosmopolitan distributions across regions, while most high-abundance ASVs were only identified in specific regions. The presence of some cosmopolitan ASVs across regions argues against dispersal limitation primarily shaping the distribution of taxa among regions. Rather, the results point to local tectonic and geologic characteristics shaping the geochemistry of continental hydrothermal systems that then select for distinct microbial assemblages. These results provide new insights into the co-evolution of hydrothermal systems and their microbial communities.

NusG is a transcription elongation factor that stimulates transcription pausing in Gram+ bacteria including B. subtilis by sequence-specific interaction with a conserved pause-inducing − 11 TTNTTT −6 motif found in the non-template DNA (ntDNA) strand within the transcription bubble. To reveal the structural basis of NusG-dependent pausing, we determined a cryo-EM structure of a paused transcription complex (PTC) containing RNA polymerase (RNAP), NusG, and the TTNTTT motif in the ntDNA strand. The interaction of NusG with the ntDNA strand rearranges the transcription bubble by positioning three consecutive T residues in a cleft between NusG and the β-lobe domain of RNAP. We revealed that the RNAP swivel module rotation (swiveling), which widens (swiveled state) and narrows (non-swiveled state) a cleft between NusG and the β-lobe, is an intrinsic motion of RNAP and is directly linked to trigger loop (TL) folding, an essential conformational change of all cellular RNAPs for the RNA synthesis reaction. We also determined cryo-EM structures of RNAP escaping from the paused transcription state. These structures revealed the NusG-dependent pausing mechanism by which NusG-ntDNA interaction inhibits the transition from swiveled to non-swiveled states, thereby preventing TL folding and RNA synthesis allosterically. This motion is also reduced by the formation of an RNA hairpin within the RNA exit channel. Thus, the pause half-life can be modulated by the strength of the NusG-ntDNA interaction and/or the stability of the RNA hairpin. NusG residues that interact with the TTNTTT motif are widely conserved in bacteria, suggesting that NusG-dependent pausing is widespread.

Transcription must be properly regulated to ensure dynamic gene expression underlying growth, development, and response to environmental cues. Regulation is imposed throughout the transcription cycle, and while many efforts have detailed the regulation of transcription initiation and early elongation, the termination phase of transcription also plays critical roles in regulating gene expression. Transcription termination can be driven by only a few proteins in each domain of life. Detailing the mechanism(s) employed provides insight into the vulnerabilities of transcription elongation complexes (TECs) that permit regulated termination to control expression of many genes and operons. Here, we describe the biochemical activities and crystal structure of the superfamily 2 helicase Eta, one of two known factors capable of disrupting archaeal transcription elongation complexes. Eta retains a twin-translocase core domain common to all superfamily 2 helicases and a well-conserved C terminus wherein individual amino acid substitutions can critically abrogate termination activities. Eta variants that perturb ATPase, helicase, single-stranded DNA and double-stranded DNA translocase and termination activities identify key regions of the C terminus of Eta that, when combined with modeling Eta-TEC interactions, provide a structural model of Eta-mediated termination guided in part by structures of Mfd and the bacterial TEC. The susceptibility of TECs to disruption by termination factors that target the upstream surface of RNA polymerase and potentially drive termination through forward translocation and allosteric mechanisms that favor opening of the clamp to release the encapsulated nucleic acids emerges as a common feature of transcription termination mechanisms.

SARS-CoV-2 proteases Mpro and PLpro are promising targets for antiviral drug development. In this study, we present an antiviral screening strategy involving a novel in-cell protease assay, antiviral and biochemical activity assessments, as well as structural determinations for rapid identification of protease inhibitors with low cytotoxicity. We identified eight compounds with anti-SARS-CoV-2 activity from a library of 64 repurposed drugs and modeled at protease active sites by in silico docking. We demonstrate that Sitagliptin and Daclatasvir inhibit PLpro, and MG-101, Lycorine HCl, and Nelfinavir mesylate inhibit Mpro of SARS-CoV-2. The X-ray crystal structure of Mpro in complex with MG-101 shows a covalent bond formation between the inhibitor and the active site Cys145 residue indicating its mechanism of inhibition is by blocking the substrate binding at the active site. Thus, we provide methods for rapid and effective screening and development of inhibitors for blocking virus polyprotein processing as SARS-CoV-2 antivirals. Additionally, we show that the combined inhibition of Mpro and PLpro is more effective in inhibiting SARS-CoV-2 and the delta variant.

Argonaute proteins are programmable nucleases that have defense and regulatory functions in both eukaryotes and prokaryotes. All known prokaryotic Argonautes (pAgos) characterized so far act on DNA targets. Here, we describe a new class of pAgos that uniquely use DNA guides to process RNA targets. The biochemical and structural analysis of Pseudooceanicola lipolyticus pAgo (PliAgo) reveals an unusual organization of the guide binding pocket that does not rely on divalent cations and the canonical set of contacts for 5'-end interactions. Unconventional interactions of PliAgo with the 5'-phosphate of guide DNA define its new position within pAgo and shift the site of target RNA cleavage in comparison with known Argonautes. The specificity for RNA over DNA is defined by ribonucleotide residues at the cleavage site. The analysed pAgos sense mismatches and modifications in the RNA target. The results broaden our understanding of prokaryotic defense systems and extend the spectrum of programmable nucleases with potential use in RNA technology.

The transcriptome-wide contributions of Rho-dependent and intrinsic (Rho-independent) transcription termination mechanisms in bacteria are unclear. By sequencing released transcripts in a wild-type strain and strains containing deficiencies in NusA, NusG and/or Rho (10 strains), we produced an atlas of terminators for the model Gram-positive bacterium Bacillus subtilis. We found that NusA and NusG stimulate 77% and 19% of all intrinsic terminators, respectively, and that both proteins participate in Rho-dependent termination. We also show that Rho stimulates termination at 10% of the intrinsic terminators in vivo. We recapitulated Rho-stimulated intrinsic termination at 5 terminators in vitro and found that Rho requires the KOW domain of NusG to stimulate this process at one of these terminators. Computational analyses of our atlas using RNAstructure, MEME suite and DiffLogo, combined with in vitro transcription experiments, revealed that Rho stimulates intrinsic terminators with weak hairpins and/or U-rich tracts by remodelling the RNA upstream of the intrinsic terminator to prevent the formation of RNA structures that could otherwise compete with the terminator hairpin. We also identified 56 putative examples of 'hybrid Rho-dependent termination', wherein classical Rho-dependent termination occurs after readthrough of a Rho-stimulated intrinsic terminator.

Tuberculosis (TB) is one of the most significant world health problems, responsible for 1.5 M deaths in 2020, and yet, current treatments rely largely on 40 year old paradigms. Although the rifamycins (RIFs), best exemplified by the drug rifampin (RMP), represent a well-studied and therapeutically effective chemotype that targets the bacterial RNA polymerase (RNAP), these agents still suffer from serious drawbacks including the following: 3-9 month treatment times; cytochrome P450 (Cyp450) induction [particularly problematic for human immunodeficiency virus-Mycobacterium tuberculosis (MTB) co-infection]; and the existence of RIF-resistant (RIFR) MTB strains. We have utilized a structure-based drug design approach to synthesize and test 15 benzoxazinorifamycins (bxRIFs), congeners of the clinical candidate rifalazil, to minimize human pregnane X receptor (hPXR) activation while improving potency against MTB. We have determined the compounds' activation of the hPXR [responsible for inducing Cyp450 3A4 (CYP3A4)]. Compound IC50s have been determined against the wild-type and the most prevalent RIFR (β-S450L) mutant MTB RNAPs. We have also determined their bactericidal activity against "normal" replicating MTB and a model for non-replicating, persister MTB. We have identified a minimal substitution and have probed larger substitutions that exhibit negligible hPXR activation (1.2-fold over the dimethyl sulfoxide control), many of which are 5- to 10-fold more potent against RNAPs and MTB than RMP. Importantly, we have analogues that are essentially equipotent against replicating MTB and non-replicating persister MTB, a property that is correlated with faster kill rates and may lead to shorter treatment durations. This work provides a proof of principle that the ansamycin core remains an attractive and effective scaffold for novel and dramatically improved RIFs.

Rifampin (RMP), a very potent inhibitor of the Mycobacterium tuberculosis (MTB) RNA polymerase (RNAP), remains a keystone in the treatment of tuberculosis since its introduction in 1965. However, rifamycins suffer from serious drawbacks, including 3- to 9-month treatment times, Cyp450 induction (particularly problematic for HIV-MTB coinfection), and resistant mutations within RNAP that yield RIF-resistant (RIFR) MTB strains. There is a clear and pressing need for improved TB therapies. We have utilized a structure-based drug design approach to synthesize and test novel benzoxazinorifamycins (bxRIF), congeners of the clinical candidate rifalazil. Our goal is to gain binding interactions that will compensate for the loss of RIF-binding affinity to the (RIFR) MTB RNAP and couple those with substitutions that we have previously found that essentially eliminate Cyp450 induction. Herein, we report a systematic exploration of 42 substituted bxRIFs that have yielded an analogue (27a) that has an excellent in vitro activity (MTB RNAP inhibition, MIC, MBC), enhanced (∼30-fold > RMP) activity against RIFR MTB RNAP, negligible hPXR activation, good mouse pharmacokinetics, and excellent activity with no observable adverse effects in an acute mouse TB model. In a time-kill study, 27a has a 7 day MBC that is ∼10-fold more potent than RMP. These results suggest that 27a may exhibit a faster kill rate than RMP, which could possibly reduce the clinical treatment time. Our synthetic protocol enabled the synthesis of ∼2 g of 27a at >95% purity in 3 months, demonstrating the feasibility of scale-up synthesis of bxRIFs for preclinical and clinical studies.

Stalling of the transcription elongation complex formed by DNA, RNA polymerase (RNAP) and RNA presents a serious obstacle to concurrent processes due to the extremely high stability of the DNA-bound polymerase. RapA, known to remove RNAP from DNA in an ATP-dependent fashion, was identified over 50 years ago as an abundant binding partner of RNAP; however, its mechanism of action remains unknown. Here, we use single-molecule magnetic trapping assays to characterize RapA activity and begin to specify its mechanism of action. We first show that stalled RNAP resides on DNA for times on the order of 10(6) seconds and that increasing positive torque on the DNA reduces this lifetime. Using stalled RNAP as a substrate we show that the RapA protein stimulates dissociation of stalled RNAP from positively supercoiled DNA but not negatively supercoiled DNA. We observe that RapA-dependent RNAP dissociation is torque-sensitive, is inhibited by GreB and depends on RNA length. We propose that stalled RNAP is dislodged from DNA by RapA via backtracking in a supercoiling- and torque-dependent manner, suggesting that RapA's activity on transcribing RNAP in vivo is responsible for resolving conflicts between converging polymerase molecular motors.

We report the effect of padding on the efficiency of the pneumatic tourniquet for the upper limb. Varying thicknesses of two commercially-available types of orthopaedic padding (Cellona and Velband) were applied to the arms of 20 volunteers, with three pressure transducers placed directly beneath the padding. A tourniquet was positioned over the padding and inflated to 220 mmHg. Significant reductions in the transmitted pressure were recorded from the transducers with both padding materials. With eight layers of padding, reductions in pressure of 13% (1% to 26%) and 18% (7% to 35%) were seen with Cellona and Velband, respectively. The reduction in pressure with Velband padding correlated with increasing arm circumference (Pearson's correlation coefficient 0.711, p < 0.001). Studies to date have examined how arm circumference affects the required tourniquet inflation pressure. Our study is the first to investigate the effect of the padding and the findings suggest that using more than two layers results in a significant reduction in the transmitted pressure.

Bacterial RNA polymerase (RNAP) is an essential multisubunit enzyme performing transcription.Regulation of this process is secured through the stage-dependent interactions of RNAP with different factors (mostly proteins).Here we report the structurefunction analysis of the functional complexes between RNAP and a unique helicase-like factor HelD [1] which is present in many Gram-positive bacteria (e.g.Bacillus subtilis and Mycobacterium smegmatis) [2, 3].HelD forms tightly bound complexes with RNAP.It simultaneously penetrates into RNAP primary and secondary channels which are responsible for nucleic acids binding and substrate delivery.HelD can also interact with the RNAP active site.Structurally, these interactions are incompatible with the binding of DNA to the RNAP core and thus with the elongation stage of transcription.This is in accordance to our functional data showing that HelD is capable of clearing RNAP of nucleic acids and that HelD can dismantle RNAP-DNA complexes.HelD itself is composed of several domains, showing structural changes in solution [2] as well as in complexes with RNAP (three different structural states obtained from the cryo-EM analysis) [3].Although we were able to link the observed dynamic behaviour with the DNA-clearing role of HelD, the recycling of HelD-bound RNAP and subsequent restart of transcription remains to be explained.HelD as well as its complexes with RNAP resisted our attempts to crystallize them for many years.In order to get to the structural details we took the advantage of recent developments in the field of single-particle cryo-EM and were able to obtain ~3Å resolution structures.The structure of HelD itself was completely unknown with no homologue in the PDB.We combined X-ray crystallography (structure of one domain) and cryo-EM, together with bioinformatics and homologous modelling and successfully built de novo a complete atomic model of the HelD protein.For the analysis of condition-dependent dynamic behaviour we used small-angle X-ray scattering [2].Results from our structural studies were supplemented with biochemical and biophysical assays (enzymology, analysis of interactions and stability) and by computational analyses [3].

RNA polymerases (RNAPs) synthesize RNA from NTPs, whereas DNA polymerases synthesize DNA from 2′dNTPs. DNA polymerases select against NTPs by using steric gates to exclude the 2′OH, but RNAPs have to employ alternative selection strategies. In single-subunit RNAPs, a conserved Tyr residue discriminates against 2′dNTPs, whereas selectivity mechanisms of multi-subunit RNAPs remain hitherto unknown. Here, we show that a conserved Arg residue uses a two-pronged strategy to select against 2′dNTPs in multi-subunit RNAPs. The conserved Arg interacts with the 2′OH group to promote NTP binding, but selectively inhibits incorporation of 2′dNTPs by interacting with their 3′OH group to favor the catalytically-inert 2′-endo conformation of the deoxyribose moiety. This deformative action is an elegant example of an active selection against a substrate that is a substructure of the correct substrate. Our findings provide important insights into the evolutionary origins of biopolymers and the design of selective inhibitors of viral RNAPs.

ABSTRACTAfter transcription termination, cellular RNA polymerases (RNAPs) are occasionally trapped on DNA, impounded in an undefined Post-Termination Complex (PTC), limiting free RNAP pool and making transcription inefficient. InEscherichia coli, a Swi2/Snf2 ATPase RapA is involved in countering such inefficiency through RNAP recycling. To understand its mechanism of RNAP recycling, we have determined the cryo-electron microscopy (cryo-EM) structures of two sets ofE. coliRapA-RNAP complexes along with RNAP core enzyme and elongation complex (EC). The structures revealed the large conformational changes of RNAP and RapA upon their association implicated in the hindrance in PTC formation. Our study reveals that although RapA binds away from the DNA binding channel of RNAP, it can close the RNAP clamp allosterically thereby preventing its non-specific DNA binding. Together with DNA binding assays, we propose that RapA acts as a guardian of RNAP by which prevents non-specific DNA binding of RNAP without affecting the sigma factor binding to RNAP core enzyme, thereby enhancing RNAP recycling.

Gene expression requires synthesis of a variety of RNAs including mRNAs, rRNAs, tRNAs and regulatory RNAs. RNA synthesis is carried out by RNA polymerase (RNAP) that proceeds with a reaction making various RNAs based on DNA sequences. Bacterial RNAP binds to a specific site of DNA, initiates RNA synthesis, and extends it until reaching a termination signal. Accuracy of transcription is maintained by high-fidelity nucleotide selection and removing misincorporated nucleotides from RNA by using the same active site of RNAP. Transcription is an essential process in bacteria, thereby making RNAP an excellent target for antimicrobial drug development.

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