Schitoviruses are widespread prokaryotic viruses that encapsidate a giant ( ~ 3500-residue) virion-associated RNA polymerase (vRNAP). During infection, vRNAP is expelled into Gram-negative bacteria, along with two additional ejection proteins, to assemble a transient DNA-ejectosome that becomes transcriptionally active, initiating viral replication. Here, we present an integrative structural analysis of the coliphage N4 vRNAP (gp50). We find that this 383 kDa enzyme is a multi-domain, single-chain RNA polymerase, structurally distinct from both compact single-chain RNAPs and large multi-subunit holoenzymes. vRNAP is composed of loosely connected domains and exhibits an intramolecular mode of allosteric regulation through its C-terminal domain. Comparative analysis of intact and genome-released virions identified gp51, which forms an outer-membrane complex, and gp52, which assembles a periplasmic tunnel. These proteins generate heterogeneous pores that facilitate the release of vRNAP. We further uncover a signaling hub in the phage tail, composed of the receptor-binding protein, tail tube, and tail plug, that detects receptor engagement and orchestrates the release of ejection proteins. We propose that the beads-on-a-string architecture of vRNAP enables the translocation of megadalton-scale protein complexes through the ~35 Å channel formed by the tail and ejection proteins. These findings establish N4 as a distinctive model for protein translocation through biological channels.

Chronic wasting disease (CWD) and scrapie are transmissible spongiform encephalopathy diseases caused by prions, infectious forms of the prion protein. Currently, immunohistochemistry (IHC) is the sole approved diagnostic method for confirming these prion infections in formalin-fixed tissues. Evaluation of prion IHC requires specially trained veterinary pathologists to assess multiple quality control parameters as well as characteristic chromogen immunolabeling patterns. This manual slide review creates a significant bottleneck for laboratories needing to rapidly scale up surveillance during periods of increased testing demand. Given the repetitive and standardized nature of prion IHC slide review, this assay represents an ideal candidate for computer-assisted diagnostics. To address this challenge, we developed a deep learning-based image analysis approach tailored to review slides from large-scale veterinary prion disease surveillance. Our training dataset included 143 prion IHC whole-slide images containing a total of 3296 annotations. Annotated images were segmented into nonoverlapping tiles and used to fine-tune a pretrained convolutional neural network, enhancing the model's ability to recognize prion-specific quality control parameters and labeling features. When tested on a separate, blinded testing dataset of 50 CWD IHC slides, the model achieved 100% concordance for chromogenic labeling when compared with evaluation by a trained veterinary pathologist. The overarching objective of this project is to automate the initial review of prion IHC slides using deep learning-based image analysis to substantially reduce the time needed for evaluation. Implementation of this technology should enhance diagnostic consistency, improve efficiency, and provide scalable capabilities essential for comprehensive prion surveillance throughout the veterinary diagnostic laboratory network.

In eukaryotes, at least three Pols (I, II, and III) are responsible for synthesizing unique RNA products. Many trans-acting factors affect the efficiency of transcription by the three Pols. Some of these factors influence more than one of the nuclear Pols. One such factor is polymerase-associated factor 1 complex (Paf1C). Paf1C, composed of five subunits in Saccharomyces cerevisiae (yeast), has been shown to promote transcription by Pols I and II and is conserved across eukaryotes. Although several studies have demonstrated that Paf1C associates with Pol I machinery, its roles in ribosomal RNA synthesis are not well-defined. In this study, we used native elongating transcript sequencing (NET-seq), to investigate the effect of the loss of two of the five Paf1C subunits (Paf1 and Cdc73) on Pol I occupancy at single-nucleotide resolution in yeast. We found that in both paf1Δ and cdc73Δ mutants, there was a significant reduction in Pol I occupancy at the 5' end of the DNA template as compared to WT yeast, accompanied by other occupancy pattern changes throughout the gene. To complement these results, we also analyzed a PRO-seq dataset that was generated with DLD1 mammalian cells. Interestingly, we found that when Paf1C was knocked-down, there was also a reduction in the occupancy of Pol I at the 5' end of the gene, consistent with our NET-seq analysis. Overall, our results support the conclusion that Paf1C is an important transcription elongation factor for Pol I and may play a conserved role across species.

At the broadest level of taxonomy, living organisms are divided into the domains Archaea, Bacteria, and Eukarya. Despite the significant differences in cellular organization, metabolic processes, and native environments between the prokaryotic Bacteria and Archaea compared to eukaryotes, the essential biological process of RNA synthesis is generally conserved across all domains. Archaea are the progenitors of Eukarya, and the homology of the singular archaeal RNA polymerase (RNAP) and eukaryotic RNA polymerases-particularly eukaryotic RNAP II (Pol II)-highlights the common evolutionary ancestry that resulted in the modern division of transcription activities between at least three distinct eukaryotic RNAPs. While detailed kinetic evaluations of the activities of bacteria and eukaryotic RNAPs have revealed both universal and significant differences in kinetic elongation schemes, identical comparisons to archaeal-derived RNAPs have largely been absent. Here, we characterize the elongation properties of Thermococcus kodakarensis (T. k.) RNAP, a model hyperthermophilic archaeon, and compare these properties to previously characterized bacterial and eukaryotic RNAPs. We demonstrate that T. k. RNAP forms transcription elongation complexes even more stable than Pol II at ambient temperatures, and remarkably stable complexes at high temperatures, wherein this archaeon thrives. We surprisingly observed no significant impact of NTP concentration on the rate of nucleotide addition by the archaeal RNAP at multiple temperatures that uniquely distinguishes the archaeal RNAP from bacterial and eukaryotic RNAPs. Our results reveal how distinct regulatory strategies can be employed for the archaeal RNAP despite the overall highly conserved structure and cellular functions of multi-subunit RNAPs. IMPORTANCE:Accurate and timely regulation of gene expression is critical for survival under dynamic conditions in all living organisms. Control of transcription initiation and elongation rates is a key parameter for cellular fitness, and determination of the conserved and unique regulatory strategies that control RNA polymerase activities is of paramount importance. How RNA synthesis is catalyzed by archaeal RNA polymerases provides insight into unique and conserved regulatory strategies for survival at the limits of life.

If the immune system is an interconnected network, then the evolution of an archetype that is ideal for fighting one pathogen should result in tradeoffs decreasing its ability to fight others. How many archetypes are there in an immune system? We infected diverse mice with Plasmodium chabaudi, and identified five distinct archetypes of responses based on the host's position in microbial load, immune activity, and host damage space. To better understand the nature of these archetypes, we developed a mathematical model of a generalized host-pathogen system. This model explains the number, and distribution of archetypes across a population of diverse hosts. Mice resilient to P. chabaudi exhibited poor outcomes when challenged with influenza, SARS-CoV-1, or Mycobacterium tuberculosis, and vice versa, supporting our tradeoff hypothesis. ### Competing Interest Statement The authors have declared no competing interest.

Prion diseases are characterized by misfolding of prion protein (PrP) from correctly folded PrPC to a disease-associated form, PrPD. Real-time quaking-induced conversion (RT-QuIC) detects prions by "seeding" reaction mixtures, which contain recombinant PrP, with samples suspected to contain prions, resulting in amplification of misfolded PrP. The assay is sensitive to inhibition by tissue constituents, including blood. Heme, a cofactor of hemoglobin (Hb), has been shown to bind PrP in an isoform-specific manner and to affect the stability of other pathogenic amyloids. Herein, tissue samples from scrapie-positive sheep were used to seed RT-QuIC reactions in the presence of heme-as free hemin, as a cofactor of Hb, and as present in whole blood. At equivalent heme concentrations, the inhibitory action of free heme was the least and that of blood the greatest, suggesting other components of Hb and whole blood have additional inhibitory actions. We also demonstrate that this inhibition of RT-QuIC acts through disruption of the recombinant PrP assay substrate, rather than destruction of PrPD seeds. Lastly, heme concentrations were measured in several ruminant tissues. Heme levels exceeded inhibitory thresholds in nearly all types of intact tissue but were reduced below inhibitory levels at a 1:1,000 dilution of most tissue types, with whole blood being one of a few notable exceptions. Our results suggest that detection of PrPD seeding activity is not precluded by exposure to heme in tissue samples, but that the final heme concentration introduced into the RT-QuIC assay mixture is the critical factor that impacts detection sensitivity. IMPORTANCE:Real-time quaking-induced conversion (RT-QuIC) is an ultrasensitive amplification assay for the detection of prions. The assay has shown exceptional performance in optimal laboratory conditions, on par with bioassay, and far surpassing current immunoassay diagnostics. However, efforts to apply RT-QuIC as a real-world diagnostic have been hampered by inconsistencies and unexpectedly low sensitivity in some field samples. This study aims to quantify and characterize the mechanism of inhibition from blood and its constituent parts, hemoglobin and heme-omnipresent components of most sample types. Such systematic evaluations of RT-QuIC inhibitory factors represent necessary steps toward the consistent and sensitive performance necessary for a field-applicable diagnostic assay.

Throughout all domains of life, RNA polymerases (Pols) synthesize RNA from DNA templates, a process called transcription. During transcription, Pols require divalent metal cations for nucleotide addition and cleavage of the nascent RNA after misincorporation or polymerase stalling. Recently, several next-generation sequencing techniques have emerged to study transcription at single-nucleotide resolution in vivo. One such technique, native elongating transcript sequencing (NET-seq), allows for isolation of transcription elongation complexes associated with a specific Pol, defining polymerase occupancy on the DNA template. Originally developed to study RNA polymerase II (Pol II), NET-seq has been adapted for RNA polymerase I (Pol I) and bacterial RNA polymerase. We recently optimized Pol I NET-seq in Saccharomyces cerevisiae, however, we omitted nucleases and their metal cofactors, which are commonly used in Pol II NET-seq. Here, we investigated the effect of CaCl2 ± MNase and MnCl2 ± DNase I on Pol I occupancy. We found that exposure of Pol I to CaCl2 and MnCl2 during NET-seq caused a significant reduction in immunoprecipitation of nascent rRNA compared to the untreated control samples, with a more severe effect when incubated with MnCl2 vs. CaCl2. Surprisingly, in contrast to the Pol I results, we found that metal treatment during Pol II NET-seq did not have a significant effect on nascent transcript capture. Taken together, these observations reinforce the conclusion that transcription elongation complexes formed by Pols I and II have unique characteristics and emphasize the need to carefully consider experimental conditions deployed in all stages of nucleic acid library generation.

Schitoviruses are widespread prokaryotic viruses that encapsidate a giant (~3,500-residue) virion-associated RNA polymerase (vRNAP). During infection, vRNAP is expelled into Gram-negative bacteria, along with two additional ejection proteins, to assemble a transient DNA-ejectosome that becomes transcriptionally active, initiating viral replication. Here, we present an integrative structural analysis of the coliphage N4 vRNAP (gp50). We find that this 383 kDa enzyme is a multi-domain, single-chain RNA polymerase, structurally distinct from both compact single-chain RNAPs and large multi-subunit holoenzymes. vRNAP is composed of loosely connected domains and exhibits an intramolecular mode of allosteric regulation through its C-terminal domain. Comparative analysis of intact and genome-released virions identified gp51, which forms an outer-membrane complex, and gp52, which assembles a periplasmic tunnel. These proteins generate heterogeneous pores that facilitate the release of vRNAP. We further uncover a signaling hub in the phage tail, composed of the receptor-binding protein, tail tube, and tail plug, that detects receptor engagement and orchestrates the release of ejection proteins. We propose that the beads-on-a-string architecture of vRNAP enables the translocation of megadalton-scale protein complexes through the ~35 Å channel formed by the tail and ejection proteins. These findings establish N4 as a distinctive model for protein translocation through biological channels.

Studies in a mouse model revealed Mycobacterium tuberculosis ( Mtb ) with a deletion of rel , regulator of the stringent response, could not establish a persistent infection. Studies in cattle with a Mycobacterium. a. paratuberculosis rel deletion mutant revealed inability to establish a persistent infection was associated with development of CD8 cytotoxic T cells (CTL) that kill intracellular bacteria. Further comparative studies ex vivo with Mbv Calmette-Guérin (BCG) and a BCG rel deletion mutant revealed no clear difference in development of CTL in vitro. As reported, a study of the recall response was conducted with cattle vaccinated with either BCG or with BCG rel, to determine if information could be obtained that would show how gene products under control of rel interfere with the CTL response to mycobacterial pathogens in vivo. The study revealed the CTL response elicited by vaccination with BCG was impaired, in comparison with the response elicited by BCG rel . Comparative analysis of the recall response ex vivo revealed the functional impairment was not associated with the timing of appearance of the recall response, expression of IFN-γ, TNF-α, IL-17, or IL-22, or molecules that mediate intracellular killing. Further studies are needed to determine how CD8 CTL functional activity is modulated in vivo by gene products regulated by rel .

RNA polymerase I (Pol I) is responsible for synthesizing ribosomal RNA, which is the rate limiting step in ribosome biogenesis. We have reported wide variability in the magnitude of the rate constants defining the rate limiting step in sequential nucleotide additions catalyzed by Pol I. in this study we sought to determine if base identity impacts the rate limiting step of nucleotide addition catalyzed by Pol I. To this end, we report a transient state kinetic interrogation of AMP, CMP, GMP, and UMP incorporations catalyzed by Pol I. We found that Pol I uses one kinetic mechanism to incorporate all nucleotides. However, we found that UMP incorporation is faster than AMP, CMP, and GMP additions. Further, we found that endonucleolytic removal of a dimer from the 3 ' end was fastest when the 3 ' terminal base is a UMP. It has been previously shown that both downstream and upstream template sequence identity impacts the kinetics of nucleotide addition. The results reported here show that the incoming base identity also impacts the magnitude of the observed rate limiting step.

Transcription is a tightly regulated, complex, and essential cellular process in all living organisms. Transcription is comprised of three steps, transcription initiation, elongation, and termination. The distinct transcription initiation and termination mechanisms of eukaryotic RNA polymerases I, II, and III (Pols I, II, and III) have long been appreciated. Recent methodological advances have empowered high-resolution investigations of the Pols’ transcription elongation mechanisms. Here, we review the kinetic similarities and differences in the individual steps of Pol I-, II-, and III-catalyzed transcription elongation, including NTP binding, bond formation, pyrophosphate release, and translocation. This review serves as an important summation of Saccharomyces cerevisiae (yeast) Pol I, II, and III kinetic investigations which reveal that transcription elongation by the Pols is governed by distinct mechanisms. Further, these studies illustrate how basic, biochemical investigations of the Pols can empower the development of chemotherapeutic compounds.

Eukaryotes express at least three nuclear DNA dependent RNA polymerases (Pols). Pols I, II, and III synthesize ribosomal (r) RNA, messenger (m) RNA, and transfer (t) RNA, respectively. Pol I and Pol III have intrinsic nuclease activity conferred by the A12.2 and C11 subunits, respectively. In contrast, Pol II requires the transcription factor (TF)IIS to confer robust nuclease activity. We recently reported that in the absence of the A12.2 subunit Pol I reverses bond formation by pyrophosphorolysis in the absence of added PPi, indicating slow PPi release. Thus, we hypothesized that Pol II, naturally lacking TFIIS, would reverse bond formation through pyrophosphorolysis. Here we report the results of transient-state kinetic experiments to examine the addition of nine nucleotides to a growing RNA chain catalyzed by Pol II. Our results indicate that Pol II reverses bond formation by pyrophosphorolysis in the absence of added PPi. We propose that, in the absence of endonuclease activity, this bond reversal may represent kinetic proofreading. Thus, given the hypothesis that Pol I evolved from Pol II through the incorporation of general transcription factors, pyrophosphorolysis may represent a more ancient form of proofreading that has been evolutionarily replaced with nuclease activity.

Tuberculosis is caused by the bacterium Mycobacterium tuberculosis (Mtb). While eukaryotic species employ several specialized RNA polymerases (Pols) to fulfill the RNA synthesis requirements of the cell, bacterial species use a single RNA polymerase (RNAP). To contribute to the foundational understanding of how Mtb and the related non-pathogenic mycobacterial species, Mycobacterium smegmatis (Msm), perform the essential function of RNA synthesis, we performed a series of in vitro transcription experiments to define the unique enzymatic properties of Mtb and Msm RNAPs. In this study, we characterize the mechanism of nucleotide addition used by these bacterial RNAPs with comparisons to previously characterized eukaryotic Pols I, II, and III. We show that Mtb RNAP and Msm RNAP demonstrate similar enzymatic properties and nucleotide addition kinetics to each other but diverge significantly from eukaryotic Pols. We also show that Mtb RNAP and Msm RNAP uniquely bind a nucleotide analog with significantly higher affinity than canonical nucleotides, in contrast to eukaryotic RNA polymerase II. This affinity for analogs may reveal a vulnerability for selective inhibition of the pathogenic bacterial enzyme. IMPORTANCE Tuberculosis, caused by the bacterium Mycobacterium tuberculosis (Mtb), remains a severe global health threat. The World Health Organization (WHO) has reported that tuberculosis is second only to COVID-19 as the most lethal infection worldwide, with more annual deaths than HIV and AIDS (WHO.int). The first-line treatment for tuberculosis, Rifampin (or Rifampicin), specifically targets the Mtb RNA polymerase. This drug has been used for decades, leading to increased numbers of multi-drug-resistant infections (Stephanie, et al). To effectively treat tuberculosis, there is an urgent need for new therapeutics that selectively target vulnerabilities of the bacteria and not the host. Characterization of the differences between Mtb enzymes and host enzymes is critical to inform these ongoing drug design efforts.

Transient state kinetic studies of eukaryotic DNA-dependent RNA polymerases (Pols) in vitro provide quantitative characterization of enzyme activity at the level of individual nucleotide addition events. Previous work revealed heterogeneity in the rate constants governing nucleotide addition by yeast RNA polymerase I (Pol I) for each position on a template DNA. In contrast, the rate constants that described nucleotide addition by yeast RNA polymerase II (Pol II) were more homogeneous. This observation led to the question, what drives the variability of rate constants governing RNA synthesis by Pol I? Are the kinetics of nucleotide addition dictated by the position of the nascent RNA within the polymerase or by the identity of the next encoded nucleotide? In this study, we examine the impact of nucleotide position (i.e. nascent RNA primer length) on the rate constants governing nine sequential nucleotide addition events catalyzed by Pol I. The results reveal a conserved trend in the observed rate constants at each position for all primer lengths used, and highlight that the 9-nucleotide, or 9-mer, RNA primer provides the fastest observed rate constants. These findings suggest that the observed heterogeneity of rate constants for RNA synthesis by Pol I in vitro is driven primarily by the template sequence.

Analysis of the recall response ex vivo in cattle vaccinated with a Mycobacterium avium subsp. paratuberculosis (Map) rel deletion mutant revealed the immune response was directed toward a 35 kD major membrane protein (MMP) of Map. Antigen presenting cells (APC) primed with MMP elicited expansion of CD8 cytotoxic memory T cells (CTL) with ability to kill intracellular bacteria. Development of CTL was MHC-restricted. The gene MAP2121c, encoding MMP, was modified for expression of MMP (tPA-MMP-2mut) in a mammalian cell line to explore the potential of developing MMP as a vaccine. Ex vivo stimulation of PBMC, from Map free cattle, with APC primed with tPA-MMP-2mut expressed p35 elicited a primary CD8 CTL response comparable to the recall response elicited with PBMC from cattle vaccinated with either the Maprel deletion mutant or MMP. In the present study, the modified gene for MMP, now referred to as p35NN, was placed into a bovine herpes virus-4 (BoHV4) vector to determine the potential use of BoHV-4AΔTK-p35NN as a peptide-based vaccine. Subcutaneous vaccination of healthy cattle with BoHV-4AΔTK-p35NN elicited a CTL recall response, as detected ex vivo. The results show use of a virus vector is an effective way for delivery of MMP as a vaccine. The immunogenic activity of MMP was not lost when modified for expression in mammalian cells. The next step is to conduct a field trial to determine if presence of an immune response to MMP prevents Map from establishing an infection.

RNA polymerase I (Pol I) is responsible for synthesizing the three largest eukaryotic ribosomal RNAs (rRNAs), which form the backbone of the ribosome. Transcription by Pol I is required for cell growth and, therefore, is subject to complex and intricate regulatory mechanisms. To accomplish this robust regulation, the cell engages a series of trans-acting transcription factors. One such factor, high mobility group protein 1 (Hmo1), has long been established as a trans-acting factor for Pol I in Saccharomyces cerevisiae; however, the mechanism by which Hmo1 promotes rRNA synthesis has not been defined. Here, we investigated the effect of the deletion of HMO1 on transcription elongation by Pol I in vivo. We determined that Hmo1 is an important activator of transcription elongation, and without this protein, Pol I accumulates across rDNA in a sequence-specific manner. Our results demonstrate that Hmo1 promotes efficient transcription elongation by rendering Pol I less sensitive to pausing in the G-rich regions of rDNA.

Saccharomyces cerevisiae is one of the most well-studied model organisms used in the scientific community. Its ease of manipulation, accessible growth conditions, short life cycle, and conserved eukaryotic metabolic pathways make it a useful model organism. Consequently, yeast has been used to investigate a myriad of phenomena, from microbial to human studies. Most of the research performed using this model organism utilizes yeast cell populations when they are growing exponentially, a growth phase aptly termed exponential or log phase. However, log phase encompasses several yeast generations and ranges several hours of yeast growth, meaning that there is a potential for variability during this "homogenous" growth phase. Cells in log phase require robust ribosome biogenesis to support their rapid growth and cell division. Interestingly, during log phase, ribosomal RNA (rRNA) synthesis (which is the first and rate limiting step in ribosome biosynthesis) has been shown to decrease prior to growth rate decline in stationary phase. In this study, we utilized several genomic and biochemical methods to elucidate the relationship between subphases of log phase and rRNA synthesis. Our results indicate that as yeast cells progress through subphases of log growth, both polymerase I transcription and rRNA processing are repressed. Overall, this study establishes a growth-phase-dependent control of rRNA synthesis that unexpectedly begins prior to the switch to stationary phase (i.e., pre-diauxic shift) as a putative mechanism of anticipating nutrient starvation.IMPORTANCESaccharomyces cerevisiae is a ubiquitously used model organism in a wide range of scientific research fields. The conventional practice when performing yeast studies is to investigate its properties during logarithmic growth phase. This growth phase is defined as the period during which the cell population doubles at regular intervals, and nutrients are not limiting. However, this growth phase lasts hours and encompasses several yeast cell generations which consequently introduce heterogeneity to log growth phase depending on their time of harvest. This study reveals significant changes in the transcriptomic landscape even in early stages of exponential growth. The overall significance of this work is the revelation that even the seemingly homogenous log growth phase is far more diverse than was previously believed.

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Eukaryotes express at least three RNA polymerases (Pols) carry out transcription, while bacteria and archaea use only one. Using transient state kinetics, we have extensively examined and compared the kinetics of both single and multi-nucleotide additions catalyzed by the three Pols. In single nucleotide addition experiments we have observed unexpected extension products beyond one incorporation, which can be attributed to misincorporation, the presence of nearly undetectable amounts of contaminating NTPs, or a mixture of the two. Here we report the development and validation of an analysis strategy to account for the presence of unexpected extension products, when they occur. Using this approach, we uncovered evidence showing that non-cognate nucleotide, thermodynamically, competes with cognate nucleotide for the active site within the elongation complex of Pol I, ΔA12 Pol I, and Pol II. This observation is unexpected because base pairing interactions provide favorable energetics for selectivity and competitive binding indicates that the affinities of cognate and non-cognate nucleotides are within an order of magnitude. Thus, we show that application of our approach will allow for the extraction of additional information that reports on the energetics of nucleotide entry and selectivity.