Structure and function of the Si3 insertion integrated into the trigger loop/helix of cyanobacterial RNA polymerase.

Cyanobacteria and evolutionarily related chloroplasts of algae and plants possess unique RNA polymerases (RNAPs) with characteristics that distinguish them 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 spanning 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.

Structure and function of the Si3 insertion integrated into the trigger loop/helix of cyanobacterial RNA polymerase.

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.

Tectonic and geological setting influence hot spring microbiology.

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).

SARS-CoV-2 polyprotein substrate regulates the stepwise M cleavage reaction.

The processing of the Coronavirus polyproteins pp1a and pp1ab by the main protease M to produce mature proteins is a crucial event in virus replication and a promising target for antiviral drug development. M cleaves polyproteins in a defined order, but how M and/or the polyproteins determine the order of cleavage remains enigmatic due to a lack of structural information about polyprotein-bound M. Here, we present the cryo-EM structures of SARS-CoV-2 M in an apo form and in complex with the nsp7-10 region of the pp1a polyprotein.

Allosteric mechanism of transcription inhibition by NusG-dependent pausing of RNA polymerase.

NusG is a transcription elongation factor that stimulates transcription pausing in Gram+ bacteria including by sequence-specific interaction with a conserved pause-inducing TTNTTT 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.

Comprehensive transcription terminator atlas for Bacillus subtilis.

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.

Programmable RNA targeting by bacterial Argonaute nucleases with unconventional guide binding and cleavage specificity.

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 structure and activities of the archaeal transcription termination factor Eta detail vulnerabilities of the transcription elongation complex.

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.

On the stability of stalled RNA polymerase and its removal by RapA.

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.

Optimization of Benzoxazinorifamycins to Improve RNA Polymerase Inhibition and Treatment of Tuberculosis.

Rifampin (RMP), a very potent inhibitor of the (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 (RIF) MTB strains. There is a clear and pressing need for improved TB therapies.

Optimization of Benzoxazinorifamycins to Minimize hPXR Activation for the Treatment of Tuberculosis and HIV Coinfection.

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- (MTB) co-infection]; and the existence of RIF-resistant (RIF) MTB strains.

Identification of SARS-CoV-2 inhibitors targeting Mpro and PLpro using in-cell-protease assay.

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.

Structural basis of RNA polymerase recycling by the Swi2/Snf2 family of ATPase RapA in Escherichia coli.

After transcription termination, cellular RNA polymerases (RNAPs) are occasionally trapped on DNA, impounded in an undefined post-termination complex (PTC), limiting the free RNAP pool and subsequently leading to inefficient transcription. In Escherichia coli, a Swi2/Snf2 family of ATPase called RapA is known to be involved in countering such inefficiency through RNAP recycling; however, the precise mechanism of this recycling is unclear. To better understand its mechanism, here we determined the structures of two sets of E.

Watching the bacterial RNA polymerase transcription reaction by time-dependent soak-trigger-freeze X-ray crystallography.

RNA polymerase (RNAP) is the central enzyme of gene expression, which transcribes DNA to RNA. All cellular organisms synthesize RNA with highly conserved multi-subunit DNA-dependent RNAPs, except mitochondrial RNA transcription, which is carried out by a single-subunit RNAP. Over 60 years of extensive research has elucidated the structures and functions of cellular RNAPs.

The mechanism of the nucleo-sugar selection by multi-subunit RNA polymerases.

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.

Structural basis of ribosomal RNA transcription regulation.

Ribosomal RNA (rRNA) is most highly expressed in rapidly growing bacteria and is drastically downregulated under stress conditions by the global transcriptional regulator DksA and the alarmone ppGpp. Here, we determined cryo-electron microscopy structures of the Escherichia coli RNA polymerase (RNAP) σ holoenzyme during rRNA promoter recognition with and without DksA/ppGpp. RNAP contacts the UP element using dimerized α subunit carboxyl-terminal domains and scrunches the template DNA with the σ finger and β' lid to select the transcription start site favorable for rapid promoter escape.

Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase.

RNA synthesis is central to life, and RNA polymerase (RNAP) depends on accessory factors for recovery from stalled states and adaptation to environmental changes. Here, we investigated the mechanism by which a helicase-like factor HelD recycles RNAP. We report a cryo-EM structure of a complex between the Mycobacterium smegmatis RNAP and HelD.

Direct binding of TFEα opens DNA binding cleft of RNA polymerase.

Opening of the DNA binding cleft of cellular RNA polymerase (RNAP) is necessary for transcription initiation but the underlying molecular mechanism is not known. Here, we report on the cryo-electron microscopy structures of the RNAP, RNAP-TFEα binary, and RNAP-TFEα-promoter DNA ternary complexes from archaea, Thermococcus kodakarensis (Tko). The structures reveal that TFEα bridges the RNAP clamp and stalk domains to open the DNA binding cleft.

Evaluation of Bacterial RNA Polymerase Inhibitors in a -Based Wound Infection Model in SKH1 Mice.

Chronic wounds infected with pathogens such as represent a worldwide health concern, especially in patients with a compromised immune system. As antimicrobial resistance has become an immense global problem, novel antibiotics are urgently needed. One strategy to overcome this threatening situation is the search for drugs targeting novel binding sites on essential and validated enzymes such as the bacterial RNA polymerase (RNAP).

Publisher Correction: Molecular basis of microhomology-mediated end-joining by purified full-length Polθ.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structural basis of reiterative transcription from the pyrG and pyrBI promoters by bacterial RNA polymerase.

Reiterative transcription is a non-canonical form of RNA synthesis by RNA polymerase in which a ribonucleotide specified by a single base in the DNA template is repetitively added to the nascent RNA transcript. We previously determined the X-ray crystal structure of the bacterial RNA polymerase engaged in reiterative transcription from the pyrG promoter, which contains eight poly-G RNA bases synthesized using three C bases in the DNA as a template and extends RNA without displacement of the promoter recognition σ factor from the core enzyme. In this study, we determined a series of transcript initiation complex structures from the pyrG promoter using soak-trigger-freeze X-ray crystallography.

Structure and function of an unusual flavodoxin from the domain .

Flavodoxins, electron transfer proteins essential for diverse metabolisms in microbes from the domain , are extensively characterized. Remarkably, although genomic annotations of flavodoxins are widespread in microbes from the domain , none have been isolated and characterized. Herein is described the structural, biochemical, and physiological characterization of an unusual flavodoxin (FldA) from , an acetate-utilizing methane-producing microbe of the domain In contrast to all flavodoxins, FldA is homodimeric, markedly less acidic, and stabilizes an anionic semiquinone.

Molecular basis of microhomology-mediated end-joining by purified full-length Polθ.

DNA polymerase θ (Polθ) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Polθ performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Polθ MMEJ of long ssDNA overhangs which model resected DSBs.

Region 3.2 of the σ factor controls the stability of rRNA promoter complexes and potentiates their repression by DksA.

The σ factor drives promoter recognition by bacterial RNA polymerase (RNAP) and is also essential for later steps of transcription initiation, including RNA priming and promoter escape. Conserved region 3.2 of the primary σ factor ('σ finger') directly contacts the template DNA strand in the open promoter complex and facilitates initiating NTP binding in the active center of RNAP.