Preprint of a response to "The Origins of SARS-CoV-2: A Critical Review" published by Holmes et al in Cell 2021 (https://doi.org/10.1016/j.cell.2021.08.017).
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ABSTRACTLambdoid 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... more
ABSTRACTLambdoid 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 translocate with RNAP as a pausing-deficient, termination-deficient Q-loaded complex. In previous work, we showed that the Q protein from 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 from 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 comple...
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Reiterative transcription initiation, observed at promoters that contain homopolymeric sequences at the transcription start site, generates RNA products having 5’ sequences non-complementary to the DNA template. Here, using... more
Reiterative transcription initiation, observed at promoters that contain homopolymeric sequences at the transcription start site, generates RNA products having 5’ sequences non-complementary to the DNA template. Here, using crystallography and cryo-EM to define structures, protein-DNA-photocrosslinking to map positions of RNAP leading and trailing edges relative to DNA, and single-molecule DNA nanomanipulation to assess RNAP-dependent DNA unwinding, we show that RNA extension in reiterative transcription initiation: (1) occurs without DNA scrunching, (2) involves a short, 2-3 bp RNA-DNA hybrid, and (3) generates RNA that exits RNAP through the portal by which scrunched nontemplate-strand DNA exits RNAP in standard transcription initiation. The results establish that, whereas RNA extension in standard transcription initiation proceeds through a scrunching mechanism, RNA extension in reiterative transcription initiation proceeds through a slippage mechanism, with slipping of RNA relat...
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Rho and NusG mediate factor-dependent transcription termination in Escherichia coli. Here, we report preparation of complexes functional in factor-dependent termination from RNA polymerase (RNAP), Rho, NusG, and synthetic nucleic-acid... more
Rho and NusG mediate factor-dependent transcription termination in Escherichia coli. Here, we report preparation of complexes functional in factor-dependent termination from RNA polymerase (RNAP), Rho, NusG, and synthetic nucleic-acid scaffolds, and we report cryo-EM structures of 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 nt of RNA interacting sequence-specifically with the exterior of Rho and 6 nt of RNA interacting sequence-specifically with the central channel of Rho, have Rho oriented relative to RNAP such that ATP-hydrolysis-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 function.One sentence summaryCryo-EM reveals the structure of the functional Rho pre-termination complex
Research Interests: RNA and RNA polymerase
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... more
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 Nu...
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Understanding how the SARS-CoV-2 virus emerged and spread to humans is an essential foundation for prioritizing future pandemic prevention and response strategies. Yet well over a year after the initial outbreak, no robust process for... more
Understanding how the SARS-CoV-2 virus emerged and spread to humans is an essential foundation for prioritizing future pandemic prevention and response strategies. Yet well over a year after the initial outbreak, no robust process for examining the origins of the pandemic has been established and critical records and biological samples that could provide essential insights into pandemic origins remain inaccessible. These shortcomings pose a significant threat to everyone and future generations. As scientists, social scientists, and science communicators, including signatories of the March 4, 2021 open letter on COVID-19 origins, we believe there is a better way forward.
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Understanding the origins of the pandemic is essential to addressing our vulnerabilities and preventing future crises. Unfortunately, as outlined in previous open letters released on March 4 and April 7, structural, procedural, and... more
Understanding the origins of the pandemic is essential to addressing our vulnerabilities and preventing future crises. Unfortunately, as outlined in previous open letters released on March 4 and April 7, structural, procedural, and analytical shortcomings of the WHO-convened joint study into COVID-19 origins have created unnecessary barriers to this understanding.
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First open letter addressed to the World Health Organisation, published on March 4, 2021.
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In 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,... more
In 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 se...
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Research Interests: Chemistry, Medicine, DNA, Escherichia coli, Metal Complexes, and 13 moreStereochemistry, Amino Acid Profile, Cysteine, Biomolecular, Base Sequence, Protein Binding, Site-directed Mutagenesis, Nucleic Acid Conformation, Chemical Modification, Biochemistry and cell biology, Solvents, Molecular Sequence Data, and Cyclic AMP receptor protein
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We report the total synthesis and configurational assignment of pargamicin A, a highly oxidized non-ribosomal peptide that potently inhibits the growth of drug-resistant bacteria. Our synthetic approach relies on late-stage piperazine... more
We report the total synthesis and configurational assignment of pargamicin A, a highly oxidized non-ribosomal peptide that potently inhibits the growth of drug-resistant bacteria. Our synthetic approach relies on late-stage piperazine ring formation and careful selection of condensation reagents to assemble the densely substituted hexapeptide backbone. This work enables the synthesis of pargamicin congeners for the development of structure-activity relationships and informs strategies to access other sterically congested piperazic acid-containing natural products.
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We report a metabolomic analysis of Streptomyces sp. ID38640, a soil isolate that produces the bacterial RNA polymerase inhibitor pseudouridimycin. The analysis was performed on the wild type, on three newly constructed and seven... more
We report a metabolomic analysis of Streptomyces sp. ID38640, a soil isolate that produces the bacterial RNA polymerase inhibitor pseudouridimycin. The analysis was performed on the wild type, on three newly constructed and seven previously reported mutant strains disabled in different genes required for pseudouridimycin biosynthesis. The results indicate that Streptomyces sp. ID38640 is able to produce, in addition to lydicamycins and deferroxiamines, as previously reported, also the lassopeptide ulleungdin, the non-ribosomal peptide antipain and the osmoprotectant ectoine. The corresponding biosynthetic gene clusters were readily identified in the strain genome. We also detected the known compound pyridindolol, for which we propose a previously unreported biosynthetic gene cluster, as well as three families of unknown metabolites. Remarkably, the levels of most metabolites varied strongly in the different mutant strains, an observation that enabled detection of metabolites unnotic...
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Transcription is the complex process in which a DNA segment is copied into a messenger RNA. Transcription can be divided in three main processes: transcription initiation, elongation and termination. In transcription initiation, RNA... more
Transcription is the complex process in which a DNA segment is copied into a messenger RNA. Transcription can be divided in three main processes: transcription initiation, elongation and termination. In transcription initiation, RNA polymerase binds to the promoter region in the DNA and gets ready to start copying the DNA. In transcription elongation, RNA polymerase moves along the DNA to create an RNA copy. In transcription termination, RNA polymerase falls off the DNA after completing the RNA copy.
Carboxamido tetramates derived from a bicyclic skeleton provide access to compounds with antibacterial activity.
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Pseudouridimycin (PUM) is a novel pseudouridine-containing peptidyl-nucleoside antibiotic that inhibits bacterial RNA polymerase (RNAP) through a binding site and mechanism different from those of clinically approved RNAP inhibitors of... more
Pseudouridimycin (PUM) is a novel pseudouridine-containing peptidyl-nucleoside antibiotic that inhibits bacterial RNA polymerase (RNAP) through a binding site and mechanism different from those of clinically approved RNAP inhibitors of the rifamycin and lipiarmycin (fidaxomicin) classes. PUM was discovered by screening microbial fermentation extracts for RNAP inhibitors. In this review, we describe the discovery and characterization of PUM. We also describe the RNAP-inhibitory and antibacterial properties of PUM. Finally, we review available information on the gene cluster and pathway for PUM biosynthesis and on the potential for discovering additional novel pseudouridine-containing nucleoside antibiotics by searching bacterial genome and metagenome sequences for sequences similar to pumJ, the pseudouridine-synthase gene of the PUM biosynthesis gene cluster.
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Transcription by RNA polymerase (RNAP) is interspersed with sequence-dependent pausing. The processes through which paused states are accessed and stabilized occur at spatiotemporal scales beyond the resolution of previous methods, and... more
Transcription by RNA polymerase (RNAP) is interspersed with sequence-dependent pausing. The processes through which paused states are accessed and stabilized occur at spatiotemporal scales beyond the resolution of previous methods, and are poorly understood. Here, we combine high-resolution optical trapping with improved data analysis methods to investigate the formation of paused states at enhanced temporal resolution. We find that pause sites reduce the forward transcription rate of nearly all RNAP molecules, rather than just affecting the subset of molecules that enter long-lived pauses. We propose that the reduced rates at pause sites allow time for the elongation complex to undergo conformational changes required to enter long-lived pauses. We also find that backtracking occurs stepwise, with states backtracked by at most one base pair forming quickly, and further backtracking occurring slowly. Finally, we find that nascent RNA structures act as modulators that either enhance o...
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We present the experimentally determined 3D structure of an intact activator-dependent transcription initiation complex comprising the Escherichia coli catabolite activator protein (CAP), RNA polymerase holoenzyme (RNAP), and a DNA... more
We present the experimentally determined 3D structure of an intact activator-dependent transcription initiation complex comprising the Escherichia coli catabolite activator protein (CAP), RNA polymerase holoenzyme (RNAP), and a DNA fragment containing positions −78 to +20 of a Class I CAP-dependent promoter with a CAP site at position −61.5 and a premelted transcription bubble. A 20-Å electron microscopy reconstruction was obtained by iterative projection-based matching of single particles visualized in carbon-sandwich negative stain and was fitted using atomic coordinate sets for CAP, RNAP, and DNA. The structure defines the organization of a Class I CAP-RNAP-promoter complex and supports previously proposed interactions of CAP with RNAP α subunit C-terminal domain (αCTD), interactions of αCTD with σ 70 region 4, interactions of CAP and RNAP with promoter DNA, and phased-DNA-bend-dependent partial wrapping of DNA around the complex. The structure also reveals the positions and shap...
Research Interests: Biophysics, Electron Microscopy, Gene regulation, Biology, RNA polymerase, and 13 moreMedicine, Multidisciplinary, Biological Sciences, DNA, Escherichia coli, Promoter, Transcription Factor, Three Dimensional, Base Sequence, D structure, Nucleic Acid Conformation, DNA fragmentation, and Molecular Sequence Data
The Escherichia coli RNA polymerase alpha-subunit binds through its carboxy-terminal domain (alpha CTD) to a recognition element, the upstream (UP) element, in certain promoters. We used genetic and biochemical techniques to identify the... more
The Escherichia coli RNA polymerase alpha-subunit binds through its carboxy-terminal domain (alpha CTD) to a recognition element, the upstream (UP) element, in certain promoters. We used genetic and biochemical techniques to identify the residues in alpha CTD important for UP-element-dependent transcription and DNA binding. These residues occur in two regions of alpha CTD, close to but distinct from, residues important for interactions with certain transcription activators. We used NMR spectroscopy to determine the secondary structure of alpha CTD, alpha CTD contains a nonstandard helix followed by four alpha-helices. The two regions of alpha CTD important for DNA binding correspond to the first alpha-helix and the loop between the third and fourth alpha-helices. The alpha CTD DNA-binding domain architecture is unlike any DNA-binding architecture identified to date, and we propose that alpha CTD has a novel mode of interaction with DNA. Our results suggest models for alpha CTD-DNA a...
Research Interests: Molecular Biology, Biology, RNA polymerase, Magnetic Resonance Spectroscopy, Medicine, and 15 moreBiological Sciences, DNA, Mutagenesis, Escherichia coli, Promoter, Genes, ribosomal RNA, DNA binding, CTD, Protein Conformation, Amino Acid Sequence, Base Sequence, Molecular Sequence Data, Psychology and Cognitive Sciences, and Medical and Health Sciences
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The catabolite activator protein (CAP) sharply bends DNA in the CAP-DNA complex, introducing a DNA kink, with a roll angle of ∼40° and a twist angle of ∼20°, between positions 6 and 7 of the DNA half-site, 5′-A1A2A3T4G5T6G7A8T9C10T11-3′... more
The catabolite activator protein (CAP) sharply bends DNA in the CAP-DNA complex, introducing a DNA kink, with a roll angle of ∼40° and a twist angle of ∼20°, between positions 6 and 7 of the DNA half-site, 5′-A1A2A3T4G5T6G7A8T9C10T11-3′ (“primary kink”). CAP recognizes the base-pair immediately 5′ to the primary-kink site, T:A6, through an “indirect-readout” mechanism involving sequence effects on the
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Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non-canonical initiating nucleotides... more
Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non-canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts, and up to ~15% NAD+ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD+ and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial transcriptional outputs, sensing NAD+ and NADH levels and adjusting transcr...
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In bacteria, transcription and translation are coupled processes, in which movement of RNA polymerase (RNAP) synthesizing mRNA is coordinated with movement of the first ribosome translating mRNA. Coupling is modulated by the transcription... more
In bacteria, transcription and translation are coupled processes, in which movement of RNA polymerase (RNAP) synthesizing mRNA is coordinated with movement of the first ribosome translating mRNA. Coupling is modulated by the transcription factors NusG--which is thought to bridge RNAP and ribosome--and NusA. Here, we report cryo-EM structures of Escherichia coli transcription-translation complexes (TTCs) containing different-length mRNA spacers between RNAP and the ribosome active-center P-site. Structures of TTCs containing short spacers show a state incompatible with NusG bridging and NusA binding (TTC-A; previously termed “expressome”). Structures of TTCs containing longer spacers reveal a new state compatible with NusG bridging and NusA binding (TTC-B) and reveal how NusG bridges and NusA binds. We propose that TTC-B mediates NusG- and NusA-dependent transcription-translation coupling.One Sentence SummaryCryo-EM defines states that mediate NusG- and NusA-dependent transcription-t...
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ABSTRACTAll organisms--bacteria, archaea, and eukaryotes--have a transcription initiation factor that contains a structural module that binds within the RNA polymerase (RNAP) active-center cleft and interacts with template-strand... more
ABSTRACTAll organisms--bacteria, archaea, and eukaryotes--have a transcription initiation factor that contains a structural module that binds within the RNA polymerase (RNAP) active-center cleft and interacts with template-strand single-stranded DNA (ssDNA) in the immediate vicinity of the RNAP active center. This transcription-initiation-factor structural module pre-organizes template-strand ssDNA to engage the RNAP active center, thereby facilitating binding of initiating nucleotides and enabling transcription initiation from initiating mononucleotides. However, this transcription-initiation-factor structural module occupies the path of nascent RNA and thus presumably must be displaced before or during initial transcription. Here, we report four sets of crystal structures of bacterial initially transcribing complexes that demonstrate, and define details of, stepwise, RNA-extension-driven displacement of the “σ finger” of the bacterial transcription initiation factor σ. The structu...
Research Interests: RNA and Multidisciplinary
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Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non-canonical initiating nucleotides... more
Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non-canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts, and up to ~10% NAD+ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD+ and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial gene expression, sensing NAD+ and NADH levels and adjusting transcriptional...
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Nucleoside-containing metabolites such as NAD can be incorporated as 5' caps on RNA by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase (RNAP). Here, we report CapZyme-seq, a... more
Nucleoside-containing metabolites such as NAD can be incorporated as 5' caps on RNA by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase (RNAP). Here, we report CapZyme-seq, a high-throughput-sequencing method that employs NCIN-decapping enzymes NudC and Rai1 to detect and quantify NCIN-capped RNA. By combining CapZyme-seq with multiplexed transcriptomics, we determine efficiencies of NAD capping by Escherichia coli RNAP for ∼16,000 promoter sequences. The results define preferred transcription start site (TSS) positions for NAD capping and define a consensus promoter sequence for NAD capping: HRRASWW (TSS underlined). By applying CapZyme-seq to E. coli total cellular RNA, we establish that sequence determinants for NCIN capping in vivo match the NAD-capping consensus defined in vitro, and we identify and quantify NCIN-capped small RNAs (sRNAs). Our findings define the promoter-sequence determinants for NCIN capping with NAD a...
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Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which kills 1.8 million annually. Mtb RNA polymerase (RNAP) is the target of the first-line antituberculosis drug rifampin (Rif). We report crystal structures of Mtb... more
Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which kills 1.8 million annually. Mtb RNA polymerase (RNAP) is the target of the first-line antituberculosis drug rifampin (Rif). We report crystal structures of Mtb RNAP, alone and in complex with Rif. The results identify an Mtb-specific structural module of Mtb RNAP and establish that Rif functions by a steric-occlusion mechanism that prevents extension of RNA. We also report novel non-Rif-related compounds--Nalpha-aroyl-N-aryl-phenylalaninamides (AAPs)--that potently and selectively inhibit Mtb RNAP and Mtb growth, and we report crystal structures of Mtb RNAP in complex with AAPs. AAPs bind to a different site on Mtb RNAP than Rif, exhibit no cross-resistance with Rif, function additively when co-administered with Rif, and suppress resistance emergence when co-administered with Rif.