High-throughput, fluorescent-aptamer-based measurements of steady-state transcription rates for the Mycobacterium tuberculosis RNA polymerase

The first step in gene expression is the transcription of DNA sequences into RNA. Regulation at the level of transcription leads to changes in steady-state concentrations of RNA transcripts, affecting the flux of downstream functions and ultimately cellular phenotypes. Changes in transcript levels are routinely followed in cellular contexts via genome-wide sequencing techniques. However, in vitro mechanistic studies of transcription have lagged with respect to throughput. Here, we describe the use of a real-time, fluorescent-aptamer-based method to quantitate steady-state transcription rates of the Mycobacterium tuberculosis RNA polymerase. We present clear controls to show that the assay specifically reports on promoter-dependent, full-length RNA transcription rates that are in good agreement with the kinetics determined by gel-resolved, α-32P NTP incorporation experiments. We illustrate how the time-dependent changes in fluorescence can be used to measure regulatory effects of nucleotide concentrations and identity, RNAP and DNA concentrations, transcription factors, and antibiotics. Our data showcase the ability to easily perform hundreds of parallel steady-state measurements across varying conditions with high precision and reproducibility to facilitate the study of the molecular mechanisms of bacterial transcription

It's about time: A synthesis of changing phenology in the Gulf of Maine ecosystem

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Staudinger, M. D., Mills, K. E., Stamieszkin, K., Record, N. R., Hudak, C. A., Allyn, A., Diamond, A., Friedland, K. D., Golet, W., Henderson, M. E., Hernandez, C. M., Huntington, T. G., Ji, R., Johnson, C. L., Johnson, D. S., Jordaan, A., Kocik, J., Li, Y., Liebman, M., Nichols, O. C., Pendleton, D., Richards, R. A., Robben, T., Thomas, A. C., Walsh, H. J., & Yakola, K. It's about time: A synthesis of changing phenology in the Gulf of Maine ecosystem. Fisheries Oceanography, 28(5), (2019): 532-566, doi: 10.1111/fog.12429.The timing of recurring biological and seasonal environmental events is changing on a global scale relative to temperature and other climate drivers. This study considers the Gulf of Maine ecosystem, a region of high social and ecological importance in the Northwest Atlantic Ocean and synthesizes current knowledge of (a) key seasonal processes, patterns, and events; (b) direct evidence for shifts in timing; (c) implications of phenological responses for linked ecological‐human systems; and (d) potential phenology‐focused adaptation strategies and actions. Twenty studies demonstrated shifts in timing of regional marine organisms and seasonal environmental events. The most common response was earlier timing, observed in spring onset, spring and winter hydrology, zooplankton abundance, occurrence of several larval fishes, and diadromous fish migrations. Later timing was documented for fall onset, reproduction and fledging in Atlantic puffins, spring and fall phytoplankton blooms, and occurrence of additional larval fishes. Changes in event duration generally increased and were detected in zooplankton peak abundance, early life history periods of macro‐invertebrates, and lobster fishery landings. Reduced duration was observed in winter–spring ice‐affected stream flows. Two studies projected phenological changes, both finding diapause duration would decrease in zooplankton under future climate scenarios. Phenological responses were species‐specific and varied depending on the environmental driver, spatial, and temporal scales evaluated. Overall, a wide range of baseline phenology and relevant modeling studies exist, yet surprisingly few document long‐term shifts. Results reveal a need for increased emphasis on phenological shifts in the Gulf of Maine and identify opportunities for future research and consideration of phenological changes in adaptation efforts.This work was supported by the Department of the Interior Northeast Climate Adaptation Science Center (G14AC00441) for MDS, AJ, and KY; the National Science Foundation's Coastal SEES Program (OCE‐1325484) for KEM, ACT, MEH, and AA; the National Aeronautics and Space Administration (NNX16 AG59G) for ACT, KEM, NRR, and KSS; the USGS Climate Research and Development Program for TGH; National Science & Engineering Research Council of Canada, University of New Brunswick, Environment Canada, Sir James Dunn Wildlife Research Centre, and New Brunswick Wildlife Trust Fund for AD. We also thank the Regional Association for Research on the Gulf of Maine for support, and the Gulf of Maine Research Institute for hosting and providing in kind resources for a two day in‐person workshop in August 2016. We greatly appreciate contributions from K. Alexander, G. Calandrino, C. Feurt, I. Mlsna, N. Rebuck, J. Seavey, and J. Sun for helping shape the initial scope of the manuscript. We thank J. Weltzin and two anonymous reviewers for their constructive comments. The contents of this paper are solely the responsibility of the authors and do not necessarily represent the views of the Northeast Climate Adaptation Science Center, U.S. Geological Survey, National Oceanographic and Atmospheric Administration, Fisheries and Oceans Canada or the US Environmental Protection Agency. This manuscript is submitted for publication with the understanding that the United States Government is authorized to reproduce and distribute reprints for Governmental purposes. None of the authors have conflicts of interest to declare in association with the contents of this manuscript

Mechanism of transcription initiation and promoter escape by E. coli RNA polymerase

To investigate roles of the discriminator and open complex (OC) lifetime in transcription initiation by Escherichia coli RNA polymerase (RNAP; α 2 ββ'ωσ 70 ), we compare productive and abortive initiation rates, short RNA distributions, and OC lifetime for the λP R and T7A1 promoters and variants with exchanged discriminators, all with the same transcribed region. The discriminator determines the OC lifetime of these promoters. Permanganate reactivity of thymines reveals that strand backbones in open regions of longlived λP R -discriminator OCs are much more tightly held than for shorter-lived T7A1-discriminator OCs. Initiation from these OCs exhibits two kinetic phases and at least two subpopulations of ternary complexes. Long RNA synthesis (constrained to be single round) occurs only in the initial phase (<10 s), at similar rates for all promoters. Less than half of OCs synthesize a full-length RNA; the majority stall after synthesizing a short RNA. Most abortive cycling occurs in the slower phase (>10 s), when stalled complexes release their short RNA and make another without escaping. In both kinetic phases, significant amounts of 8-nt and 10-nt transcripts are produced by longer-lived, λP R -discriminator OCs, whereas no RNA longer than 7 nt is produced by shorter-lived T7A1-discriminator OCs. These observations and the lack of abortive RNA in initiation from short-lived ribosomal promoter OCs are well described by a quantitative model in which ∼1.0 kcal/mol of scrunching free energy is generated per translocation step of RNA synthesis to overcome OC stability and drive escape. The different length-distributions of abortive RNAs released from OCs with different lifetimes likely play regulatory roles. RNA polymerase | open complex lifetime | transcription initiation | abortive RNA | hybrid length M any facets of transcription initiation by E. coli RNA polymerase (RNAP; α 2 ββ′ωσ 70 ) have been elucidated, but significant questions remain about the mechanism or mechanisms by which initial transcribing complexes (ITC) with a short RNA-DNA hybrid decide to advance and escape from the promoter to enter elongation mode, or, alternately, to stall, release their short RNA, and reinitiate (abortive cycling). For RNAP to escape, its sequencespecific interactions with promoter DNA in the binary open complex (OC) must be overcome. The open regions of promoter DNA in the binary OC are the −10 region (six residues, with specific interactions between σ 2.2 and the nontemplate strand), the discriminator region (typically six to eight residues with no consensus sequence, the upstream end of which interacts with σ 1.2 ), and the transcription start site (TSS, +1) and adjacent residue (+2), which are in the active site of RNAP What drives promoter escape? Escape involves disrupting all the favorable interactions involved in forming and stabilizing the binary OC as well as σ-core interactions. Escape from these interactions is fundamentally driven by the favorable chemical (free) energy change of RNA synthesis, but this energy must be stored in the ITC in each step before escape. Proposed means of energy storage as the length of the RNA-DNA hybrid increases include the stresses introduced by scrunching distortions of the discriminator regions of the open strands in the cleft (2, 5, 6) and by unfavorable interactions of the RNA-DNA hybrid with the hairpin loop of σ 3.2 (7-10). Scrunching of the discriminator region of the template strand is proposed to be most significant for Significance The enzyme RNA polymerase (RNAP) transcribes DNA genetic information into RNA. Regulation of transcription occurs largely in initiation; these regulatory mechanisms must be understood. Lifetimes of transcription-capable RNAP-promoter open complexes (OCs) vary greatly, dictated largely by the DNA discriminator region, but the significance of OC lifetime for regulation was unknown. We observe that a significantly longer RNA:DNA hybrid is synthesized before RNAP escapes from long-lived λP R -promoter OCs as compared with shorter-lived T7A1 promoter OCs. We quantify the free energy needed to overcome OC stability and allow escape from the promoter and elongation of the nascent RNA, and thereby predict escape points for ribosomal (rrnB P1) and lacUV5 promoters. Longer-lived OCs produce longer abortive RNAs, which likely have specific regulatory roles

Mechanism of transcription initiation and promoter escape by E. coli RNA polymerase

To investigate roles of the discriminator and open complex (OC) lifetime in transcription initiation by Escherichia coli RNA polymerase (RNAP; α 2 ββ'ωσ 70 ), we compare productive and abortive initiation rates, short RNA distributions, and OC lifetime for the λP R and T7A1 promoters and variants with exchanged discriminators, all with the same transcribed region. The discriminator determines the OC lifetime of these promoters. Permanganate reactivity of thymines reveals that strand backbones in open regions of longlived λP R -discriminator OCs are much more tightly held than for shorter-lived T7A1-discriminator OCs. Initiation from these OCs exhibits two kinetic phases and at least two subpopulations of ternary complexes. Long RNA synthesis (constrained to be single round) occurs only in the initial phase (<10 s), at similar rates for all promoters. Less than half of OCs synthesize a full-length RNA; the majority stall after synthesizing a short RNA. Most abortive cycling occurs in the slower phase (>10 s), when stalled complexes release their short RNA and make another without escaping. In both kinetic phases, significant amounts of 8-nt and 10-nt transcripts are produced by longer-lived, λP R -discriminator OCs, whereas no RNA longer than 7 nt is produced by shorter-lived T7A1-discriminator OCs. These observations and the lack of abortive RNA in initiation from short-lived ribosomal promoter OCs are well described by a quantitative model in which ∼1.0 kcal/mol of scrunching free energy is generated per translocation step of RNA synthesis to overcome OC stability and drive escape. The different length-distributions of abortive RNAs released from OCs with different lifetimes likely play regulatory roles. RNA polymerase | open complex lifetime | transcription initiation | abortive RNA | hybrid length M any facets of transcription initiation by E. coli RNA polymerase (RNAP; α 2 ββ′ωσ 70 ) have been elucidated, but significant questions remain about the mechanism or mechanisms by which initial transcribing complexes (ITC) with a short RNA-DNA hybrid decide to advance and escape from the promoter to enter elongation mode, or, alternately, to stall, release their short RNA, and reinitiate (abortive cycling). For RNAP to escape, its sequencespecific interactions with promoter DNA in the binary open complex (OC) must be overcome. The open regions of promoter DNA in the binary OC are the −10 region (six residues, with specific interactions between σ 2.2 and the nontemplate strand), the discriminator region (typically six to eight residues with no consensus sequence, the upstream end of which interacts with σ 1.2 ), and the transcription start site (TSS, +1) and adjacent residue (+2), which are in the active site of RNAP What drives promoter escape? Escape involves disrupting all the favorable interactions involved in forming and stabilizing the binary OC as well as σ-core interactions. Escape from these interactions is fundamentally driven by the favorable chemical (free) energy change of RNA synthesis, but this energy must be stored in the ITC in each step before escape. Proposed means of energy storage as the length of the RNA-DNA hybrid increases include the stresses introduced by scrunching distortions of the discriminator regions of the open strands in the cleft (2, 5, 6) and by unfavorable interactions of the RNA-DNA hybrid with the hairpin loop of σ 3.2 (7-10). Scrunching of the discriminator region of the template strand is proposed to be most significant for Significance The enzyme RNA polymerase (RNAP) transcribes DNA genetic information into RNA. Regulation of transcription occurs largely in initiation; these regulatory mechanisms must be understood. Lifetimes of transcription-capable RNAP-promoter open complexes (OCs) vary greatly, dictated largely by the DNA discriminator region, but the significance of OC lifetime for regulation was unknown. We observe that a significantly longer RNA:DNA hybrid is synthesized before RNAP escapes from long-lived λP R -promoter OCs as compared with shorter-lived T7A1 promoter OCs. We quantify the free energy needed to overcome OC stability and allow escape from the promoter and elongation of the nascent RNA, and thereby predict escape points for ribosomal (rrnB P1) and lacUV5 promoters. Longer-lived OCs produce longer abortive RNAs, which likely have specific regulatory roles

Genome Sequence of the Model Mushroom Schizophyllum Commune

Much remains to be learned about the biology of mushroom-forming fungi, which are an important source of food, secondary metabolites and industrial enzymes. The wood-degrading fungus Schizophyllum commune is both a genetically tractable model for studying mushroom development and a likely source of enzymes capable of efficient degradation of lignocellulosic biomass. Comparative analyses of its 38.5-megabase genome, which encodes 13,210 predicted genes, reveal the species\u27s unique wood-degrading machinery. One-third of the 471 genes predicted to encode transcription factors are differentially expressed during sexual development of S. commune. Whereas inactivation of one of these, fst4, prevented mushroom formation, inactivation of another, fst3, resulted in more, albeit smaller, mushrooms than in the wild-type fungus. Antisense transcripts may also have a role in the formation of fruiting bodies. Better insight into the mechanisms underlying mushroom formation should affect commercial production of mushrooms and their industrial use for producing enzymes and pharmaceuticals

1968

Maintenance of Golf Carts by Thomas Pepe (page 1) Why a Golf Course Superintendent Should Play Golf by Stephen Skowronski (2) Tree Pruning by Martin Walsh (3) Golf Course Labor: A Dilemma by Robert Barber (5) Turf Problems by Alexander M. Radko (A-1) 1967 Turfgrass Problems by Lee Record (A-3) Southern Turfgrass Production and Problems by Ralph W. White Jr. (A-5) Canadian Turf Grass Production and Problems by David Moote (A-8) Turf Research Abroad by C.R. Skogley (A-13) Turf Research at Home by Victor B. Younger (A-14) Turfgrass Research - An Industrial Approach by J. A. Simmons (A-16) Cutting Labor Costs in Turfgrass Managemnt by Tom Mascaro (A-24) The Reluctant human by John W. Denison (A-28) The Problem Drinker - Management Responsibility by G.E. Osburn (A-31) Contemporary Design Standards by Geoffrey S. Cornish (A-34) Construction - Superintendents\u27 Viewpoint by Robert E. Grant (A-36) Construction b Contract and the Role of the Superintendent by David Canavan (A-39) Seed Production by Robert J. Peterson (A-41) Cemetery Turf Maintenance on a High and Low Budget by Stanley Sosenski (A-45) Ten Steps to a Good Lawn by John Zak (A-49) Review of Herbicides for Turf Weed Control by Alvin A. Baber (A-53

Genetic landscape of congenital insensitivity to pain and hereditary sensory and autonomic neuropathies

Congenital insensitivity to pain (CIP) and hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders exclusively or predominantly affecting the sensory and autonomic neurons. Due to the rarity of the diseases and findings based mainly on single case reports or small case series, knowledge about these disorders is limited. Here, we describe the molecular workup of a large international cohort of CIP/HSAN patients including patients from normally under-represented countries. We identify 80 previously unreported pathogenic or likely pathogenic variants in a total of 73 families in the >20 known CIP/HSAN-associated genes. The data expand the spectrum of disease-relevant alterations in CIP/HSAN, including novel variants in previously rarely recognized entities such as ATL3-, FLVCR1- and NGF-associated neuropathies and previously under-recognized mutation types such as larger deletions. In silico predictions, heterologous expression studies, segregation analyses and metabolic tests helped to overcome limitations of current variant classification schemes that often fail to categorize a variant as disease-related or benign. The study sheds light on the genetic causes and disease-relevant changes within individual genes in CIP/HSAN. This is becoming increasingly important with emerging clinical trials investigating subtype or gene-specific treatment strategies