Mechanism of Transcription Termination
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Recent Developments in Terminator Technology in Saccharomyces Cerevisiae
Journal of Bioscience and Bioengineering VOL. 128 No. 6, 655e661, 2019 www.elsevier.com/locate/jbiosc REVIEW Recent developments in terminator technology in Saccharomyces cerevisiae Takashi Matsuyama1 Toyota Central R&D Lab, Nagakute, Aichi 480-1192, Japan Received 20 March 2019; accepted 7 June 2019 Available online 16 July 2019 Metabolically engineered microorganisms that produce useful organic compounds will be helpful for realizing a sustainable society. The budding yeast Saccharomyces cerevisiae has high utility as a metabolic engineering platform because of its high fermentation ability, non-pathogenicity, and ease of handling. When producing yeast strains that produce exogenous compounds, it is a prerequisite to control the expression of exogenous enzyme-encoding genes. Terminator region in a gene expression cassette, as well as promoter region, could be used to improve metabolically engineered yeasts by increasing or decreasing the expression of the target enzyme-encoding genes. The findings on terminators have grown rapidly in the last decade, so an overview of these findings should provide a foothold for new developments. Ó 2019, The Society for Biotechnology, Japan. All rights reserved. [Key words: Terminator; Metabolic engineering; Genetic switch; Gene expression; Synthetic biology; Saccharomyces cerevisiae] In order to realize a sustainable society, many studies have been metabolic engineering, (iv) terminator selection for optimization of conducted to develop microorganisms that efficiently produce gene expression, (v) use of terminators as genetic switches, (vi) useful organic compounds using metabolic engineering (1). mechanism of action of highly active terminators and (vii) chal- Saccharomyces cerevisiae is considered to have high utility as a lenges in artificial terminator development. platform for these genetically-modified microorganisms for rea- sons such as high fermentation ability, high safety and easy BASIC FUNCTIONS OF THE TERMINATOR handling (2). -
Structural and Mechanistic Studies of the THI Box and SMK Box Riboswitches
Structural and Mechanistic Studies of the THI Box and SMK Box Riboswitches Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Angela Mae Smith, M.S. Graduate Program in Microbiology The Ohio State University 2009 Dissertation Committee: Professor Tina Henkin, Advisor Professor Kurt Fredrick Professor Michael Ibba Professor Ross Dalbey ABSTRACT Organisms have evolved a variety of mechanisms for regulating gene expression. Expression of individual genes is carefully modulated during different stages of cell development and in response to changing environmental conditions. A number of regulatory mechanisms involve structural elements within messenger RNAs (mRNAs) that, in response to an environmental signal, undergo a conformational change that affects expression of a gene encoded on that mRNA. RNA elements of this type that operate independently of proteins or translating ribosomes are termed riboswitches. In this work, the THI box and SMK box riboswitches were investigated in order to gain insight into the structural basis for ligand recognition and the mechanism of regulation employed by each of these RNAs. Both riboswitches are predicted to regulate at the level of translation initiation using a mechanism in which the Shine-Dalgarno (SD) sequence is occluded in response to ligand binding. For the THI box riboswitch, the studies presented here demonstrated that 30S ribosomal subunit binding at the SD region decreases in the presence of thiamin pyrophosphate (TPP). Mutation of conserved residues in the ligand binding domain resulted in loss of TPP-dependent repression in vivo. Based on these experiments two classes of mutant phenotypes were identified. -
A Region of Phage Lambda Required for Antitermination of Transcription
Cell, Vol. 31, 61-70, November 1982, Copyright 0 1982 by MIT Analysis of nutR; A Region of Phage Lambda Required for Antitermination of Transcription Eric R. Olson, Eric L. Flamm* and 1971; Friedman and Baron, 1974; Keppel et al., David I. Friedman 1974; Friedman et al., 1976, 1981; Greenblatt et al., Department of Microbiology and Immunology 1980). The sites include putative sites of pN action, The University of Michigan Medical School called nut (Salstrom and Szybalski, 1978; Rosenberg Ann Arbor, Michigan 48109 et al., 1978), as well as termination signals (Roberts, 1969; Rosenberg et al., 1978; Rosenberg and Court, 1979). Summary Consideration of the nature of the various factors involved in pN action formed the basis for the following The N gene product of coliphage lambda acts with model of pN action (discussed by Friedman and host factors (Nus) through sites (not) to render Gottesman, 1982). Transcription initiating at the early subsequent downstream transcription resistant to promoters PR and P, extends through the n&R and a variety of termination signals. These sites, not!? nutl sites, respectively (Figure 1). At these sites RNA and nutL, are downstream, respectively, from the polymerase is modified, rendering continuing tran- early promoters PR and PL. Thus a complicated set scription resistant to termination signals. The nature of molecular interactions are likely to occur at the of the promoter appears to play no role in pN action, nut sites. We have selected mutations in the nutR since placement of the n&R region downstream from region that reduce the effectiveness of pN in alter- the bacterial gal operon promoter results in termina- ing transcription initiating at the PR promoter. -
A Framework for Automated Enrichment of Functionally Significant Inverted Repeats in Whole Genomes
Missouri University of Science and Technology Scholars' Mine Computer Science Faculty Research & Creative Works Computer Science 01 Feb 2010 A Framework for Automated Enrichment of Functionally Significant Inverted Repeats in Whole Genomes Cyriac Kandoth Fikret Ercaļ Missouri University of Science and Technology, [email protected] Ronald L. Frank Missouri University of Science and Technology, [email protected] Follow this and additional works at: https://scholarsmine.mst.edu/comsci_facwork Part of the Biology Commons, and the Computer Sciences Commons Recommended Citation C. Kandoth et al., "A Framework for Automated Enrichment of Functionally Significant Inverted Repeats in Whole Genomes," BMC Bioinformatics, vol. 11, no. SUPPL. 6, BioMed Central Ltd., Feb 2010. The definitive version is available at https://doi.org/10.1186/1471-2105-11-S6-S20 This Article - Conference proceedings is brought to you for free and open access by Scholars' Mine. It has been accepted for inclusion in Computer Science Faculty Research & Creative Works by an authorized administrator of Scholars' Mine. This work is protected by U. S. Copyright Law. Unauthorized use including reproduction for redistribution requires the permission of the copyright holder. For more information, please contact [email protected]. Kandoth et al. BMC Bioinformatics 2010, 11(Suppl 6):S20 http://www.biomedcentral.com/1471-2105/11/S6/S20 PROCEEDINGS Open Access A framework for automated enrichment of functionally significant inverted repeats in whole genomes Cyriac Kandoth1*, Fikret Ercal1†, Ronald L Frank2† From Seventh Annual MCBIOS Conference. Bioinformatics: Systems, Biology, Informatics and Computation Jonesboro, AR, USA. 19-20 February 2010 Abstract Background: RNA transcripts from genomic sequences showing dyad symmetry typically adopt hairpin-like, cloverleaf, or similar structures that act as recognition sites for proteins. -
Definition of RNA Polymerase II Cotc Terminator Elements in the Human
Cell Reports Article Definition of RNA Polymerase II CoTC Terminator Elements in the Human Genome Takayuki Nojima,1 Martin Dienstbier,2 Shona Murphy,1 Nicholas J. Proudfoot,1,* and Michael J. Dye1,* 1Sir William Dunn School of Pathology, University of Oxford, South Parks Road, OX1 3RE Oxford, UK 2MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, OX1 3QX Oxford, UK *Correspondence: [email protected] (N.J.P.), [email protected] (M.J.D.) http://dx.doi.org/10.1016/j.celrep.2013.03.012 SUMMARY ing and is entirely dependent upon the presence of a functional poly(A) signal (Whitelaw and Proudfoot, 1986; Connelly and Mammalian RNA polymerase II (Pol II) transcription Manley, 1988). Pre-mRNA cleavage at the poly(A) site, mediated termination is an essential step in protein-coding by the 30 end processing complex (Shi et al., 2009), generates 0 gene expression that is mediated by pre-mRNA pro- two RNA products; a 5 cleavage product that is stabilized by polyadenylation as it is processed into mRNA and a 30 cleavage cessing activities and DNA-encoded terminator ele- 0 0 ments. Although much is known about the role of product that is subject to rapid degradation by the 5 -3 exonu- pre-mRNA processing in termination, our under- clease Xrn2. Such Xrn2-mediated transcript degradation has been shown to have a role in Pol II termination (West et al., standing of the characteristics and generality of 2004). The above could lead one to conclude that the poly(A) terminator elements is limited. -
Terminator Operon Reporter: Combining a Transcription Termination Switch with Reporter Technology for Improved Gene Synthesis and Synthetic Biology Applications
www.nature.com/scientificreports OPEN Terminator Operon Reporter: combining a transcription termination switch with reporter Received: 02 March 2016 Accepted: 04 May 2016 technology for improved gene Published: 25 May 2016 synthesis and synthetic biology applications Massimiliano Zampini1, Luis A. J. Mur1, Pauline Rees Stevens1, Justin A. Pachebat1, C. James Newbold1, Finbarr Hayes2 & Alison Kingston-Smith1 Synthetic biology is characterized by the development of novel and powerful DNA fabrication methods and by the application of engineering principles to biology. The current study describes Terminator Operon Reporter (TOR), a new gene assembly technology based on the conditional activation of a reporter gene in response to sequence errors occurring at the assembly stage of the synthetic element. These errors are monitored by a transcription terminator that is placed between the synthetic gene and reporter gene. Switching of this terminator between active and inactive states dictates the transcription status of the downstream reporter gene to provide a rapid and facile readout of the accuracy of synthetic assembly. Designed specifically and uniquely for the synthesis of protein coding genes in bacteria, TOR allows the rapid and cost-effective fabrication of synthetic constructs by employing oligonucleotides at the most basic purification level (desalted) and without the need for costly and time-consuming post-synthesis correction methods. Thus, TOR streamlines gene assembly approaches, which are central to the future development of synthetic biology. Synthetic biology is an emerging discipline that aims to apply engineering principles to biology and has the poten- tial to drive a revolution in biotechnology1. The discipline has progressed particularly as a result of the implemen- tation of novel and powerful DNA construction techniques that allow for efficient assembly and manipulation of sequences both in vitro and in vivo2,3. -
DNA Sequence; Operator; Repressor; Regulatory Region; Dyad Symmetry; Plasmid Pbr322; Sl Nuclease Mapping)
Gene, 23 (1983) 149-156 149 Elsevier Overlapping divergent promoters control expression of TnlO tetracycline resistance (DNA sequence; operator; repressor; regulatory region; dyad symmetry; plasmid pBR322; Sl nuclease mapping) Kevin P. Bertrand *, Kathleen Posh, Lewis V. Wray Jr. ** and William S. Reznikoff ** Department of Microbiology, University of California, Irvine, CA 92717 (U.S.A.) Tel. (714) 833- 6115, and ** Depart- ment of Biochemistry, University of Wisconsin, Madison, WI 53706 (U.S.A.) Tel. (608) 262 - 3608 (Received February 7th, 1983) (Accpeted February l&h, 1983) SUMMARY We have previously examined the genetic organization and regulation of the TnZO tetracycline-resistance determinant in Escherichia coli K-12. The structural genes for retA, the TnlO tetracycline-resistance function, and for tetR, the TnlO lef repressor, are transcribed in opposite directions from promoters in a regulatory region located between the two structural genes. Expression of both t&I and t&R is induced by tetracycline. Here we report the DNA sequence of the TnlO tet regulatory region. The locations of the tetA and tetR promoters within this region were defined by Sl nuclease mapping of the 5’ ends of in vivo tet RNA. The t&4 and tetR promoters overlap; the transcription start points are separated by 36 bp. We propose that two similar regions of dyad symmetry within the TnlO tet regulatory region are operator sites at which tet repressor binds to tet DNA, thereby in~biting transcription initiation at the ietA and tetR promoters. The TnlO ret regulatory region and the pBR322 tet regulatory region show significant DNA sequence homology (53%). -
The Regulatory Region of the Divergent Argecbh Operon in Escherichia Coli K-12
Volume 10 Number 24 1982 Nucleic Acids Research The regulatory region of the divergent argECBH operon in Escherichia coli K-12 Jacques Piette*, Raymond Cunin*, Anne Boyen*, Daniel Charlier*, Marjolaine Crabeel*, Franpoise Van Vliet*, Nicolas Glansdorff*, Craig Squires+ and Catherine L.Squires + *Nficrobiology, Vrije Universiteit Brussel, and Research Institute of the CERIA, 1, Ave. E. Giyson, B-1070 Brussels, Belgium, and +Department of Biological Sciences, Columbia University, New York, NY 10027, USA Received 24 September 1982; Revised and Accepted 24 November 1982 ABSTRACT The nucleotide sequence of the control region of the divergent argECBH operon has been established in the wild type and in mutants affecting expression of these genes. The argE and argCBH promoters face each other and overlap with an operator region containing two domains which may act as distinct repressor binding sites. A long leader sequence - not involved in attenuation - precedes argCBH. Overlapping of the argCBH promoter and the region involved in ribosome mobilization for argE translation explains the dual effect of some mutations. Mutations causing semi-constitutive expression of argE improve putative promoter sequences within argC. Implications of these results regarding control mechanisms in amino acid biosynthesis and their evolution are discussed. INTRODUCTION Divergently transcribed groups of functionally related genes are not exceptional in Escherichia coli (1-10). In only a few instances, however, are the sites responsible for the expression and the regulation of the flanking genes organized in an inte- grated fashion such that the gene cluster constitutes a bipolar operon, with an internal operator region flanked by promoters facing each other. The argECBH cluster (Fig.1) is one of the earliest reported examples of such a pattern. -
Mutational Analysis of a Yeast Transcriptional Terminator (Linker Substitution/Saccharomyces Cerevisiae) BRIAN I
Proc. Natl. Acad. Sci. USA Vol. 86, pp. 4097-4101, June 1989 Cell Biology Mutational analysis of a yeast transcriptional terminator (linker substitution/Saccharomyces cerevisiae) BRIAN I. OSBORNE AND LEONARD GUARENTE Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 Communicated by S. E. Luria, January 23, 1989 ABSTRACT We have isolated and mutagenized a DNA scribed and 3' ends for both mRNAs fall within the 83-bp fragment from Saccharomyces cerevisiae that specifies mRNA region (Fig. 1). 3' end formation for the convergently transcribed CYCI and We showed previously that transcription of CYCJ-lacZ on UTRI genes. An in vivo plasmid supercoiling assay previously a plasmid in S. cerevisiae resulted in an increase in negative showed that this fragment is a transcriptional terminator, and supercoiling of the plasmid (27). By inserting the terminator "run-on" assays shown here are consistent with this interpre- fragment into various positions in CYCI-lacZ, we showed tation. The poly(A) sites in the mRNAs formed by the fragment that truncated transcripts of the expected sizes were gener- are the same whether the fragment resides at the native location ated, showing that the terminator fragment was sufficient to or at a heterologous location. No single linker substitution generate 3' ends. Further, the degree of relative, negative abolishes the fragment's activity, whereas certain large, supercoiling ofthese plasmids was proportional to the lengths nonoverlapping deletions have strong, deleterious effects. of truncated transcripts. We interpreted these changes in Therefore, the yeast terminator behaves more like rho- negative supercoiling as resulting from changes in the num- dependent bacterial terminators than terminators of higher bers of transcribing RNA polymerase molecules, suggesting eukaryotes. -
Facilitated Recycling Pathway for RNA Polymerase III
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Cell, Vol. 84, 245±252, January 26, 1996, Copyright 1996 by Cell Press Facilitated Recycling Pathway for RNA Polymerase III Giorgio Dieci* and Andre Sentenac exception of TFIID, and a new complex must be reas- Service de Biochimie et Ge ne tique Mole culaire sembled for each new cycle (Zawel et al., 1995). TFIID Commissariat aÁ l'Energie Atomique±Saclay remains promoter-bound through the transcription cycle F-91191 Gif-sur-Yvette Cedex and therefore facilitates reinitiation on the same tem- France plate (Hawley and Roeder, 1987; Jiang and Gralla, 1993). However, the release and reassociation of the other fac- tors make the reinitiation process more complex than Summary in the case of pol III and thus increase the number of critical checkpoints for transcriptional modulation. We show that the high in vitro transcription efficiency Some activators of pol II transcription have indeed been of yeast RNA pol III is mainly due to rapid recycling. shown to be required at each new cycle (Arnosti et al., Kinetic analysis shows that RNA polymerase recycling 1993; Roberts et al., 1995). Reinitiation on class III genes, on preassembled tDNA·TFIIIC·TFIIIB complexes is which bypasses almost all the steps required for the much faster than the initial transcription cycle. High initial transcription cycle, does not allow such a fine efficiency of RNA pol III recycling is favored at high regulation, but potentially leads to more efficient RNA UTP concentrations and requires termination at the production. -
Novel Mechanisms of Transcriptional Regulation by Leukemia Fusion Proteins
Novel mechanisms of transcriptional regulation by leukemia fusion proteins A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirement for the degree of Doctor of Philosophy in the Department of Cancer and Cell Biology of the College of Medicine by Chien-Hung Gow M.S. Columbia University, New York M.D. Our Lady of Fatima University B.S. National Yang Ming University Dissertation Committee: Jinsong Zhang, Ph.D. Robert Brackenbury, Ph.D. Sohaib Khan, Ph.D. (Chair) Peter Stambrook, Ph.D. Song-Tao Liu, Ph.D. ABSTRACT Transcription factors and chromatin structure are master regulators of homeostasis during hematopoiesis. Regulatory genes for each stage of hematopoiesis are activated or silenced in a precise, finely tuned manner. Many leukemia fusion proteins are produced by chromosomal translocations that interrupt important transcription factors and disrupt these regulatory processes. Leukemia fusion proteins E2A-Pbx1 and AML1-ETO involve normal function transcription factor E2A, resulting in two distinct types of leukemia: E2A-Pbx1 t(1;19) acute lymphoblastic leukemia (ALL) and AML1-ETO t(8;21) acute myeloid leukemia (AML). E2A, a member of the E-protein family of transcription factors, is a key regulator in hematopoiesis that recruits coactivators or corepressors in a mutually exclusive fashion to regulate its direct target genes. In t(1;19) ALL, the E2A portion of E2A-Pbx1 mediates a robust transcriptional activation; however, the transcriptional activity of wild-type E2A is silenced by high levels of corepressors, such as the AML1-ETO fusion protein in t(8;21) AML and ETO-2 in hematopoietic cells. -
Characterization of Transcriptional Control Elements in Cluster E Mycobacteriophage Ukulele
The University of Maine DigitalCommons@UMaine Honors College Spring 5-2016 Characterization of Transcriptional Control Elements in Cluster E Mycobacteriophage Ukulele Campbell Belisle Haley University of Maine Follow this and additional works at: https://digitalcommons.library.umaine.edu/honors Part of the Biochemistry Commons, and the Spanish Linguistics Commons Recommended Citation Haley, Campbell Belisle, "Characterization of Transcriptional Control Elements in Cluster E Mycobacteriophage Ukulele" (2016). Honors College. 397. https://digitalcommons.library.umaine.edu/honors/397 This Honors Thesis is brought to you for free and open access by DigitalCommons@UMaine. It has been accepted for inclusion in Honors College by an authorized administrator of DigitalCommons@UMaine. For more information, please contact [email protected]. CHARACTERIZATION OF TRANSCRIPTIONAL CONTROL ELEMENTS IN CLUSTER E MYCOBACTERIOPHAGE UKULELE By Campbell Belisle Haley Thesis Submitted in Partial Fulfillment of the Requirements for a Degree in Honors (Biochemistry and Spanish) The Honors College University of Maine May 2016 Advisory Committee: Sally D. Molloy, Advisor, Assistant Professor, Honors College and Department of Molecular and Biomedical Sciences Keith W. Hutchison, Professor Emeritus, Department of Molecular and Biomedical Sciences Melissa Maginnis, Assistant Professor, Department of Molecular and Biomedical Sciences Dorothy Croall, Professor, Department of Molecular and Biomedical Sciences David Gross, Adjunct Associate Professor in Honors Abstract Mycobacteriophage (phage) are a diverse group of viruses that infect Mycobacterium. Their study allows further understanding of viral evolution and genetics. Phage tightly control gene expression and transcribe their genes using host RNA polymerases. This project identifies potential transcriptional control elements in the genome of mycobacteriophage Ukulele. Promoters are sequences of the genome that allow binding of RNA polymerase and initiation of transcription.