Regulation of transcription elongation via interactions of RNA polymerase with sequence elements and accessory factors

RNA polymerase (RNAP) is the enzyme responsible for catalyzing the first step in a cascade of events leading towards gene expression. Since the dissemination of genetic information plays such a critical and integral role within the context of cellular processes, RNAP must uphold the stringent guidelines of catalyzing processive DNA-directed synthesis of RNA transcripts at a reasonable rate and with high fidelity. To ensure that these requirements are met and sustained, RNAP serves as a major target of rigorous regulation and is consequently subject to extrinsic regulatory factors, such as transcription accessory proteins, and to regulatory sequence elements, such as pause and termination sites located throughout the genome. Chapter 2 explores the ability of RNAP to recognize a terminally misincorporated base in the nascent RNA transcript and to cleave that base from the transcript. Additionally, the effects of GreA and GreB, transcript cleavage factors, on RNAP containing terminally misincorporated transcript are also investigated. My results indicate that RNAP does not preferentially cleave a misincorporated base. In fact, the GreB-mediated cleavage of terminally correct and terminally incorrect bases is indistinguishable. I conclude that, in the event of misincorporation, decay into the activated pathway serves as a signal to recruit GreA and GreB. Through Gre-mediated action, RNAP may backtrack along the nascent transcript and cleave the segment of RNA containing the error. Chapter 3 studies the role of downstream elements in RNAP in regulating transcription elongation. RNAPs harboring mutations in two regions (Walker B motif and fork loop 2) composing the allosteric NTP binding site are studied for their ability to recognize pause and termination sites. I demonstrate that the Walker B motif is important for the recognition of pause and termination sites. The recognition of termination signals by the Walker B motif is also important in vivo. Additionally, I propose that the formation of two classes of pause states does not proceed through a common slow intermediate, as previously thought. Chapter 4 presents the development of method by which the interaction of a transcription accessory factor with a stalled elongation complex can be probed. This method utilizes the technique of through-prism total internal reflection fluorescence (TIRF) microscopy. I present single-molecule data that permit me to estimate the lifetime and the dissociation constant of NusG

SPIRE Point Source Catalog Explanatory Supplement

The Spectral and Photometric Imaging Receiver (SPIRE) was launched as one of the scientific instruments on board of the space observatory Herschel. The SPIRE photometer opened up an entirely new window in the Submillimeter domain for large scale mapping, that up to then was very difficult to observe. There are already several catalogs that were produced by individual Herschel science projects. Yet, we estimate that the objects of only a fraction of these maps will ever be systematically extracted and published by the science teams that originally proposed the observations. The SPIRE instrument performed its standard photometric observations in an optically very stable configuration, only moving the telescope across the sky, with variations in its configuration parameters limited to scan speed and sampling rate. This and the scarcity of features in the data that require special processing steps made this dataset very attractive for producing an expert reduced catalog of point sources that is being described in this document. The Catalog was extracted from a total of 6878 unmodified SPIRE scan map observations. The photometry was obtained by a systematic and homogeneous source extraction procedure, followed by a rigorous quality check that emphasized reliability over completeness. Having to exclude regions affected by strong Galactic emission, that pushed the limits of the four source extraction methods that were used, this catalog is aimed primarily at the extragalactic community. The result can serve as a pathfinder for ALMA and other Submillimeter and Far-Infrared facilities. 1,693,718 sources are included in the final catalog, splitting into 950688, 524734, 218296 objects for the 250\mu m, 350\mu m, and 500\mu m bands, respectively. The catalog comes with well characterized environments, reliability, completeness, and accuracies, that single programs typically cannot provide

A Defective mRNA Cleavage and Polyadenylation Complex Facilitates Expansions of Transcribed (GAA) n Repeats Associated with Friedreich’s Ataxia

Expansions of microsatellite repeats are responsible for numerous hereditary diseases in humans, including myotonic dystrophy and Friedreich's ataxia. Whereas the length of an expandable repeat is the main factor determining disease inheritance, recent data point to genomic trans modifiers that can impact the likelihood of expansions and disease progression. Detection of these modifiers may lead to understanding and treating repeat expansion diseases. Here, we describe a method for the rapid, genome-wide identification of trans modifiers for repeat expansion in a yeast experimental system. Using this method, we found that missense mutations in the endoribonuclease subunit (Ysh1) of the mRNA cleavage and polyadenylation complex dramatically increase the rate of (GAA) n repeat expansions but only when they are actively transcribed. These expansions correlate with slower transcription elongation caused by the ysh1 mutation. These results reveal an interplay between RNA processing and repeat-mediated genome instability, confirming the validity of our approach. Keywords: genome instability; repeat expansion; RNA polyadenylation; RNA processing; transcription-replication conflicts; Friedreich’s ataxia; DNA double-strand breaks; trans-modifiers of repeat expansions; genetic screen; whole-genome sequencin

Collaboration With Deaf Communities to Conduct Accessible Health Surveillance

Introduction Populations of deaf sign language users experience health disparities unmeasured by current public health surveillance. Population-specific health data are necessary to collaboratively identify health priorities and evaluate interventions. Standardized, reproducible, and language-concordant data collection in sign language is impossible via written or telephone surveys. Methods Deaf and hearing researchers, community members, and other stakeholders developed a broad computer-based health survey based on the telephone-administered Behavioral Risk Factor Surveillance System. They translated survey items from English to sign language, evaluated the translations, and filmed the survey items for inclusion in their custom software. They initiated the second Rochester Deaf Health Survey in 2013 (n=211). Analyses (conducted in 2015) compared Rochester Deaf Health Survey 2013 findings with those of the Behavioral Risk Factor Surveillance System with the general adult population in the same community (2012, n=1,816). Results The Rochester Deaf Health Survey 2013 participants’ mean age was 44.7 (range, 18—87) years. Most were deaf since birth or early childhood (87.1%) and highly educated (53.6% with ≥4 years of college). The median household income was \u3c $35,000. The prevalence of current smokers was low (8.1%). Nearly all (93.8%) reported having health insurance, yet barriers to appropriate health care were evident, with high emergency department use (16.2% with two or more past-year visits) and 22.7% forgoing needed health care in the past year because of cost. Conclusions Community-engaged research with deaf populations identifies strengths and priorities, providing essential information otherwise missing from existing public health surveillance, and forming a foundation for collaborative dissemination to facilitate broader inclusion of deaf communities

Examining the role of parental self-regulation in family physical activity: a mixed-methods approach.

Physical activity (PA) is essential for good health. However, parents risk becoming less active because of the demands of parenting. This has consequences for children as parents are role models. The present study used a mixed-methods approach to explore parental self-regulation associated with PA. Data were collected from 36 parents with preschool-aged children. They were interviewed about their PA and their family's PA. Parents also completed PA and self-regulation questionnaires and wore an accelerometer for five days. Qualitative data were examined using an inductive approach to thematic analysis. It showed that parents felt that they had limited time for personal PA. Mothers' self-regulation was driven by an ethic of care and subjective norms, whereas fathers' self-regulation was driven by beliefs about the importance of autonomy. Nevertheless, both parents saw caring for their children as the main priority. Quantitative data were examined using multiple regression analyses. Results showed that different self-regulatory behaviours predicted the PA of mothers and fathers. Which predictors were significant depended on the type of activity and how it was measured. The findings warrant longitudinal research that would enable the effect of family dynamics on self-regulation associated with PA to be assessed

The effects of cocoa supplementation, caloric restriction, and regular exercise, on oxidative stress markers of brain and memory in the rat model

The effects of treadmill running (8 weeks, 5 times/week, 1h/day at 27 m/min), caloric restriction, and cocoa supplementation on brain function and oxidative stress markers were tested. The Morris maze test was used to appraise rat memory. Regular exercise significantly improved spatial learning performance. The level of oxidative stress was measured by the concentration of carbonylated proteins. The free radical concentration increased in brain of the training groups but not the controls. The content of reactive carbonyl derivates did not change with exercise, suggesting that the increased production of reactive oxygen species (ROS) were well tolerated in this experimental model. Caloric restriction (CR) decreased the accumulation of free radicals in the frontal lobe. The protein content of brain-derived neutrophic factors (BDNFs) was evaluated and changes did not occur either with exercise or cocoa supplementation treatments. These data did not show significant effects of the administration of cocoa (2% w/w) on the concentration of ROS, BDNF or on spatial memory. Conversely, exercise and CR can play a role in ROS generation and brain function

Microneedle Array Design Determines the Induction of Protective Memory CD8+ T Cell Responses Induced by a Recombinant Live Malaria Vaccine in Mice

BACKGROUND: Vaccine delivery into the skin has received renewed interest due to ease of access to the immune system and microvasculature, however the stratum corneum (SC), must be breached for successful vaccination. This has been achieved by removing the SC by abrasion or scarification or by delivering the vaccine intradermally (ID) with traditional needle-and-syringes or with long microneedle devices. Microneedle patch-based transdermal vaccine studies have predominantly focused on antibody induction by inactivated or subunit vaccines. Here, our principal aim is to determine if the design of a microneedle patch affects the CD8(+) T cell responses to a malaria antigen induced by a live vaccine. METHODOLOGY AND FINDINGS: Recombinant modified vaccinia virus Ankara (MVA) expressing a malaria antigen was percutaneously administered to mice using a range of silicon microneedle patches, termed ImmuPatch, that differed in microneedle height, density, patch area and total pore volume. We demonstrate that microneedle arrays that have small total pore volumes induce a significantly greater proportion of central memory T cells that vigorously expand to secondary immunization. Microneedle-mediated vaccine priming induced significantly greater T cell immunity post-boost and equivalent protection against malaria challenge compared to ID vaccination. Notably, unlike ID administration, ImmuPatch-mediated vaccination did not induce inflammatory responses at the site of immunization or in draining lymph nodes. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that the design of microneedle patches significantly influences the magnitude and memory of vaccine-induced CD8(+) T cell responses and can be optimised for the induction of desired immune responses. Furthermore, ImmuPatch-mediated delivery may be of benefit to reducing unwanted vaccine reactogenicity. In addition to the advantages of low cost and lack of pain, the development of optimised microneedle array designs for the induction of T cell responses by live vaccines aids the development of solutions to current obstacles of immunization programmes