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.
View Article and Find Full Text PDFTuberculosis is caused by the bacterium (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, (Msm), perform the essential function of RNA synthesis, we performed a series of transcription experiments to define the unique enzymatic properties of Mtb and Msm RNAPs.
View Article and Find Full Text PDFEukaryotes 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.
View Article and Find Full Text PDFAnalysis 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.
View Article and Find Full Text PDFRNA 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.
View Article and Find Full Text PDFChronic wasting disease (CWD) is a fatal prion disease of cervids spreading across North America. More effective mitigation efforts may require expansion of the available toolkit to include new methods that provide earlier antemortem detection, higher throughput, and less expense than current immunohistochemistry (IHC) methods. The rectal mucosa near the rectoanal junction is a site of early accumulation of CWD prions and is safely sampled in living animals by pinch biopsy.
View Article and Find Full Text PDFEukaryotes 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.
View Article and Find Full Text PDFPrions or prion-like aggregates such as those composed of PrP, α-synuclein, and tau are key features of proteinopathies such as prion, Parkinson's and Alzheimer's diseases, respectively. Their presence on solid surfaces may be biohazardous under some circumstances. PrP prions bound to solids are detectable by ultrasensitive real-time quaking-induced conversion (RT-QuIC) assays if the solids can be immersed in assay wells or the prions transferred to pads.
View Article and Find Full Text PDFGenes (Basel)
February 2024
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.
View Article and Find Full Text PDFTranscription 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.
View Article and Find Full Text PDFRibosome biogenesis is a vital and highly energy-consuming cellular function occurring primarily in the nucleolus. Cancer cells have an elevated demand for ribosomes to sustain continuous proliferation. This study evaluated the impact of existing anticancer drugs on the nucleolus by screening a library of anticancer compounds for drugs that induce nucleolar stress.
View Article and Find Full Text PDFTransient 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.
View Article and Find Full Text PDFB-cell leukemia is a rare form of hematologic neoplasia in sheep, especially in adult animals. We present a case report of a 5-year-old WhiteFace Sheep wether with suspected acute lymphoblastic leukemia. The patient, a second-generation relative of ewes experimentally inoculated with atypical scrapie, exhibited acute lethargy and loss of appetite.
View Article and Find Full Text PDFAn 8-year-old Saanen goat doe was seen for inappetence, tachycardia, and intermittent bluish-grey discoloration of the oral mucous membranes. On physical examination, the goat was mildly tachypneic and tachycardic, with reduced sounds auscultated on the left side of the thorax. Euthanasia was elected.
View Article and Find Full Text PDFCancer cells are especially sensitive to perturbations in ribosome biogenesis as they rely on finely tuned protein homeostasis to facilitate their rapid growth and proliferation. While ribosome synthesis and cancer have a well-established relationship, ribosome biogenesis has only recently drawn interest as a cancer therapeutic target. In this study, we exploited the relationship between ribosome biogenesis and cancer cell proliferation by using a potent ribosome biogenesis inhibitor, RBI2 (Ribosome Biogenesis Inhibitor 2), to perturb cancer cell growth and viability.
View Article and Find Full Text PDFRNA polymerase I (Pol I) synthesizes ribosomal RNA (rRNA), which is the first and rate-limiting step in ribosome biosynthesis. A12.2 (A12) is a critical subunit of Pol I that is responsible for activating Pol I's exonuclease activity.
View Article and Find Full Text PDFRNA Polymerase I (Pol I) has recently been recognized as a cancer therapeutic target. The activity of this enzyme is essential for ribosome biogenesis and is universally activated in cancers. The enzymatic activity of this multi-subunit complex resides in its catalytic core composed of RPA194, RPA135, and RPA12, a subunit with functions in RNA cleavage, transcription initiation and elongation.
View Article and Find Full Text PDFChronic wasting disease (CWD) continues to spread in wild and farmed cervid populations. Early antemortem CWD testing of farmed cervids is of considerable interest to producers and regulatory agencies as a tool to combat this spread. The tissues accessible for antemortem sampling are limited and include biopsy of the tonsil and recto-anal mucosa-associated lymphoid tissue (RAMALT).
View Article and Find Full Text PDFHere we present an optimized protocol for monitoring and analyzing single nucleotide incorporation by RNA polymerases. This protocol describes the assembly of Saccharomyces cerevisiae RNA polymerase I elongation complexes in a promoter-independent system in vitro. We describe how to collect a time course using a quench-flow, a rapid mixing instrument, and subsequently resolve reactions on a polyacrylamide gel.
View Article and Find Full Text PDFEukaryotic RNA polymerase II (Pol II) is an essential enzyme that lies at the core of eukaryotic biology. Due to its pivotal role in gene expression, Pol II has been subjected to a substantial number of investigations. We aim to further our understanding of Pol II nucleotide incorporation by utilizing transient-state kinetic techniques to examine Pol II single nucleotide addition on the millisecond time scale.
View Article and Find Full Text PDFCancer cells require robust ribosome biogenesis to maintain rapid cell growth during tumorigenesis. Because RNA polymerase I (Pol I) transcription of the ribosomal DNA (rDNA) is the first and rate-limiting step of ribosome biogenesis, it has emerged as a promising anti-cancer target. Over the last decade, novel cancer therapeutics targeting Pol I have progressed to clinical trials.
View Article and Find Full Text PDFOne of the first steps in ribosome biogenesis is transcription of the ribosomal DNA by RNA polymerase I (Pol I). Processing of the resultant rRNA begins cotranscriptionally, and perturbation of Pol I transcription elongation results in defective rRNA processing. Mechanistic insight regarding the link between transcription elongation and ribosome assembly is lacking because of limited in vivo methods to assay Pol I transcription.
View Article and Find Full Text PDFRNA Polymerase I (Pol I) synthesizes rRNA, which is the first and rate-limiting step in ribosome biogenesis. Factors governing the stability of the polymerase complex are not known. Previous studies characterizing Pol I inhibitor BMH-21 revealed a transcriptional stress-dependent pathway for degradation of the largest subunit of Pol I, RPA194.
View Article and Find Full Text PDFEukaryotes express three nuclear RNA polymerases (Pols I, II, and III) that are essential for cell survival. Despite extensive investigation of the three Pols, significant knowledge gaps regarding their biochemical properties remain because each Pol has been evaluated independently under disparate experimental conditions and methodologies. To advance our understanding of the Pols, we employed identical transcription assays for direct comparison of their elongation rates, elongation complex (EC) stabilities, and fidelities.
View Article and Find Full Text PDFmiR-486 is a muscle-enriched microRNA, or "myomiR," that has reduced expression correlated with Duchenne muscular dystrophy (DMD). To determine the function of miR-486 in normal and dystrophin-deficient muscles and elucidate miR-486 target transcripts in skeletal muscle, we characterized knockout mice ( KO). KO mice developed disrupted myofiber architecture, decreased myofiber size, decreased locomotor activity, increased cardiac fibrosis, and metabolic defects were exacerbated in KO: (DKO) mice.
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