2 Outline 1. Principles of Transcriptional Regulation 2. Regulation of Transcription Initiation: Lac Operon 3. The Case of Phage λ: Layers of Regulation 2
4 Part 1: Principles of Transcription Regulation 4
5 Fig 7-1 Activation by recruitment of RNA polymerase RNA polymerase a. Absence of Regulatory Proteins: Basal level b. Repressor binding to the operator represses expression Repressor No transcription c. Activator binding activates expression Activator activated level 5
6 Fig 7-2 Allosteric activation of RNA polymerase No transcription Activator activated level Allostery is a mechanism by which activators interact with the stable closed complex and induce a conformational change that causes transition to the open complex. 6
7 Action at a Distance and DNA Looping Fig 7-3 Interaction between proteins and DNA 7
8 Fig 7-4 DNA-bending protein can facilitate interaction between DNA-binding proteins at a distance 8
9 [Watch the animation-regulation of the transcription initiation!] Apply your knowledge! 9
11 Part 2: Regulation of Transcription Initiation : Lac Operon 11
12 Control element Structural genes Fig 7-5 Operon 12
13 Operon: a unit of prokaryotic gene expression and regulation which typically includes: 1. Structural genes for enzymes in a specific biosynthetic and metabolic pathway whose expression is coordinately controlled. 2. Control elements, such as operator sequence. 3. Regulator gene(s) whose products recognize the control elements. These genes are usually transcribed from a different promoter. 13
14 Fig 7-6 Lactose operon The enzymes encoded by lacz, lacy, laca 14 are transcribed at a high level only when lactose is available as the sole carbon source.
15 The lacz, lacy, laca genes are transcribed into a single laczya mrna (polycistronic mrna) under the control of a single promoter P lac. lacz lacy codes for β-galactosidase ( 半乳糖苷酶 ) for lactose hydrolysis encodes a cell membrane protein called lactose permease to transport lactose across the cell wall laca encodes a thiogalactoside transacetylase to get rid of the toxic thiogalacosides 15
16 An activator and a repressor together control the Lac operon expression The activator: CAP (Catabolite Activator Protein) or CRP (camp Receptor Protein); responding to the glucose level. The repressor: lac repressor that is encoded by LacI gene; responding to the lactose. 16
17 Fig 7-7 Expression of the lac genes RNA polymerase Glu Lac Basal level No transcription activated level 17
18 Fig 7-8 The control region of the lac operon 18
19 Fig 7-9 Activation of the lac promoter by CAP a CTD: C-terminal domain of the a subunit of RNAP 19
20 Fig 7-10 Footprinting method for detecting protein-binding site in DNA *--Radioactive labels --cutting sites by DNase --Protein, e.g., Lac repressor Footprint
21 *--Radioactive labels --Protein Fig 7-11 Gel mobility shift assay for identifying proteins that bind DNA
22 CAP and Lac repressor bind DNA using a common structural motif: helix-turn-helix motif Fig 7-12 Binding of a protein with a helixturn-helix domain to DNA 22
23 Lac operon contains three operators: the primary operator and two other operators located 400 bp downstream and 90 bp upstream, respectively. DNA looping Fig 7-13 Lac repressor binds as a tetramer to two operators 23
24 Combinatorial Control : CAP controls other genes as well. A regulator (CAP) works together with different repressors at different genes, this is an example of Combinatorial Control. In fact, CAP acts at more than 100 genes in E.coli, working with an array of partners. 24
25 Part 3: The Case of Bacteriophage l: Layers of Regulation 25
26 Bacteriophage l is a virus that infects E. coli. Upon infection, the phage can propagate in either of two ways : lytically or lysogenically. Fig 7-14 Growth and induction of l lysogen ( 溶原性细菌 ) Bacterial genome l genome Infection induction lytic ( 溶菌性 ) growth l repressor lysogenic( 溶原性 ) growth New phage
27 Recombination proteins of phage Phage DNA replication proteins Lysis proteins The phage l has a 50- kb genome and ~50 genes. Fig 7-15 Map of phage l Head genes Tail genes
28 Fig. 7-16: Transcription in the l control regions in lytic and lysogenic growth lytic lysogenic P R and P L : rightward and leftward promoter, respectively P RM : Promoter for repressor maintenance
29 The ci gene encodes l repressor, which can both activate and repress transcription As a repressor, it binds to sites that overlap the promoter and excludes RNA polymerase As an activator, it works like CAP by recruitment. Fig l repressor
30 Cro (for control of repressor and other things) only represses transcription. It is a single domain protein that binds as a dimer to 17-bp DNA sequences.
31 Fig. 18 Relative positions of promoter and operator sites in O R ci P RM P R cro O R3 O R2 O R1 There are 6 operators in the right (3) and left (3) control regions of bacteriophage l. l repressor and Cro can each bind to any one of six operators, but with dramatically different affinity. l repressor binds O R1 most easily while Cro binds O R3 with the highest affinity.
32 Fig Layer (1): Cooperative binding of l repressor to DNA Monomer Without cooperativity, a tenfold higher concerntration of repressor would be needed to bind O R2. Dimer Tetramer Not bound Low affinity High affinity
33 Fig Cooperative binding reaction Cooperative binding of a repressor to its operator sites. Binding of a protein to a single site
34 Fig Layer (2): l Repressor and Cro bind in different patterns to control lytic and lysogenic growth Lysogen ci l Repressor cro Lytic growth ci O R3 O R2 O R1 induction cro cro P RM P R
35 1. DNA damage activates RecA in E. coli 2. RecA stimulates l repressor to undergo autocleavage, resulting in the removal the C- terminal domain and the immediate loss of dimerization and binding cooperativity. 3. Repressor dissociates from O R1 -O R2 & O R1 -O R2. Loss of repression triggers transcription from P R and P L, leading to lytic growth. Fig Layer (3): Lytic induction requires proteolytic cleavage of l repressor lytic ci cro lysogenic P L ci P RM P R cro
36 For induction to work efficiently, the level of l repressor in a lysogen must be tightly regulated. Layer (4): Keep it not too low by positive autoregulation: l repressor binding at O R2 activates its own transcription from P RM. Layer (5): Keep it not too high by negative autoregulation: when the repressor level goes too high, it will bind to O R3 as well, which will prevent transcription from P RM. 36
37 Fig Interaction of l repressors at O R and O L Negative autoregulation of repressor requires long-distance interactions and a large DNA loop: cooperative binding at O R3 and O L3 to prevent the synthesis of new l repressor.
38 Fig Layer (6): Another activator, l cii, controls the decision between lytic and lysogenic growth upon infection of a new host. cii is transcribed from P R and ciii is transcribed from P L. cii protein is a transcriptional activator that binds to P RE and stimulates the transcription of ci gene (l repressor).
39 Fig Establishment of lysogeny
40 Establishment of lysogeny 1. P R and P L are constitutive promoters that promote transcription once the phage enter the cells. 2. P R directs the synthesis of both Cro and cii proteins. Cro favors lytic development while cii favors lysogenic growth by activating the synthesis of l repressor. 3. The efficiency with which cii directs transcription of ci gene (l repressor) is critical in deciding the lysogeny. 40
41 Growth conditions of E. coli control the stability of cii protein and thus the lytic/lysogenic choice. --cii is degraded by a specific protease FtsH. --Layer (7): If growth conditions is good, cii is unstable, repressor is not made, and the phage tend to grow lytically. --Layer (8): When conditions are poor for bacterial growth, cii becomes stable, repressor accumulates, and lysogens form. --cii are stabilized by ciii, which serves as an alternative substrate for FtsH.
42 Layer (9): Transcriptional Antitermination in l development --Two l phage regulatory proteins N and Q are called antiterminators. --The transcripts controlled by l N and Q proteins terminate a few hundred to a thousand nucleotides downstream of the promoter in the absence of N and Q proteins. --l N and Q proteins prevent the termination at some termination sites by regulating RNA polymerase and promote the transcription of the early and late genes for the lytic growth of the phage.
43 Fig Binding sites for proteins N and Q N protein binds to the RNA transcribed from DNA containing a nut sequence, while Q protein binds to the QBE DNA site 43
44 Layer (10): Retroregulation : An interplay of controls on RNA synthesis and stability determines int gene expression The Int protein is the enzyme that integrates the phage genome into that of the host cell during formation of lysogen. The cii protein activates the promoter P I that directs expression of the int gene as well as the promoter P RE responsible for repressor synthesis. The int gene is transcribed from both P L and P I, but the int mrna initiated at P L is degraded by cellular nucleases.
45 Site of termination in absence of N protein Int gene Transcribed from P I in the absence of N protein, resistant to degradation Fig Transcribed from P L in the presence of N protein, going beyond the terminator and targeted for degradation
46 Summary for 10-layer regulations in phage l (1) Cooperative binding of l repressor to DNA at O R2. (2) l Repressor and Cro bind in different patterns to control lytic and lysogenic growth. (3) Upon DNA damage, proteolytic cleavage of l repressor. (4) Positive autoregulation: l repressor binding at O R2 (5) Negative autoregulation: cooperative binding of repressor at O R3 and O L3. (6) cii protein stimulates the transcription of ci gene (encoding l repressor ) in a new host. (7) cii degradation by a specific protease FtsH under good growth conditions. (8) cii stabilization by ciii, another substrate for FtsH. (9) l N and Q proteins prevent the transcriptional termination of the degradable Int mrna. (10)Retroregulation : targeted degradation of Int mrna for lytic growth.
47 Summary of Chapter 7 1. Principles of gene regulation. Activator, repressor, operator, recruitment, allostery. 2. Regulation of transcription initiation in bacteria: the Lac operon. The activator: CAP The repressor: Lac repressor 3. The case of phage l 10 layers of regulation l repressor and Cro and their binding; proteolytic cleavage of l repressor (lytic induction); autoregulation; control of the decision to lytic or lysogenic growth by l cii; antiterminators; retroregulation of Int gene. 47
What makes cells different from each other? How do cells respond to information from environment? Regulation of: - Transcription - prokaryotes - eukaryotes - mrna splicing - mrna localisation and translation
Module 3 Questions Section 1. Essay and Short Answers. Use diagrams wherever possible 1. With the use of a diagram, provide an overview of the general regulation strategies available to a bacterial cell.
Bacterial and Phage Genetic Switches Prof. C. J. Dorman Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin. Lecture 1 The genetic switch controlling the lytic-lysogen
Gene Transcription in Prokaryotes Operons: in prokaryotes, genes that encode protein participating in a common pathway are organized together. This group of genes, arranged in tandem, is called an OPERON.
RNA Structure, Function, and Synthesis RNA RNA differs from DNA in both structural and functional respects. RNA has two major structural differences: each of the ribose rings contains a 2 -hydroxyl, and
MICROBIAL GENETICS Gene Regulation: The Operons Pradeep Kumar Burma Reader Department of Genetics University of Delhi South Campus Benito Juarez Road New Delhi-110021 E-mail: firstname.lastname@example.org 05-May-2006
Gene Regulation -- The Lac Operon Specific proteins are present in different tissues and some appear only at certain times during development. All cells of a higher organism have the full set of genes:
Chapter 18 Regulation of Gene Expression 18.1. Gene Regulation Is Necessary By switching genes off when they are not needed, cells can prevent resources from being wasted. There should be natural selection
Transcription in prokaryotes Elongation and termination After initiation the σ factor leaves the scene. Core polymerase is conducting the elongation of the chain. The core polymerase contains main nucleotide
AP * BIOLOGY GENE REGULATION Teacher Packet AP* is a trademark of the College Entrance Examination Board. The College Entrance Examination Board was not involved in the production of this material. Pictures
STO-143 Gene Switches Teacher Information Summary Kit contains How do bacteria turn on and turn off genes? Students model the action of the lac operon that regulates the expression of genes essential for
Chem 465 Biochemistry II Name: 2 points Multiple choice (4 points apiece): 1. Formation of the ribosomal initiation complex for bacterial protein synthesis does not require: A) EF-Tu. B) formylmethionyl
AP Biology Review Packet 4: Viruses, Bacteria and Expression & DNA Technology 3A1- DNA, and in some cases RNA, is the primary source of heritable information. 3B1- Gene Regulation results in differential
PYF7 3/21/05 7:59 PM Page 105 Chapter 7 acteriophage acteriophage or phage for short are viruses that infect only bacteria. In contrast to cells that grow from an increase in the number of their components
Chapter 25 DNA Metabolism Multiple Choice Questions 1. DNA replication Page: 977 Difficulty: 2 Ans: C The Meselson-Stahl experiment established that: A) DNA polymerase has a crucial role in DNA synthesis.
Alison Stewart 11/12/06 Prokaryotic Cells, Eukaryotic cells and HIV: Structures, Transcription and Transport Section Handout Discussion Week #7 Compare and contrast the organization of eukaryotic, prokaryotic
Dynamics of Biological Systems Part I - Biological background and mathematical modelling Paolo Milazzo (Università di Pisa) Dynamics of biological systems 1 / 53 Introduction The recent developments in
IP-Free E. coli Inducible Expression Vectors E. coli expression vectors are available with the following promoters: T5 or T7 (IPTG-inducible), rhabad (rhamnose-inducible), ara (arabinose and IPTG-inducible)
Lecture 36: Basics of DNA Cloning-II Note: Before starting this lecture students should have completed Lecture 35 Sequential steps involved in DNA cloning using plasmid DNA as vector: Molecular cloning
Lab Exercise: Transformation Background Genetic transformation is used in many areas of biotechnology, and, at its heart, requires two things: Donor DNA and recipient cells. Cells which receive the donor
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 39, Issue of September 28, pp. 36168 36173, 2001 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Neural Model
LECTURE PRESENTATIONS For CAMPBELL BIOLOGY, NINTH EDITION Jane B. Reece, Lisa A. Urry, Michael L. Cain, Steven A. Wasserman, Peter V. Minorsky, Robert B. Jackson Chapter 18 Regulation of Gene Expression
Chapter 8 Microbial Genetics Structure and Function of the Genetic Material Chromosomes are cellular structures made up of genes that carry hereditary information. Genetics is the study of how genes carry
AP BIOLOGY 2009 SCORING GUIDELINES Question 4 The flow of genetic information from DNA to protein in eukaryotic cells is called the central dogma of biology. (a) Explain the role of each of the following
JOURNAL OF BACTERIOLOGY, Jan. 2007, p. 298 304 Vol. 189, No. 2 0021-9193/07/$08.00 0 doi:10.1128/jb.01215-06 MINIREVIEW A New Look at Bacteriophage Genetic Networks Donald L. Court, 1 * Amos B. Oppenheim,
DNA CLONING - What is cloning? The isolation of discrete pieces of DNA from their host organism and their amplification through propagation in the same or a different host More recently an alternitive,
Chapter 13B: Animal Viruses 1. Overview of Animal Viruses 2. DNA Viruses 3. RNA Viruses 4. Prions 1. Overview of Animal Viruses Life Cycle of Animal Viruses The basic life cycle stages of animal viruses
Bacterial Transformation and Plasmid Purification Chapter 5: Background History of Transformation and Plasmids Bacterial methods of DNA transfer Transformation: when bacteria take up DNA from their environment
An Application of Model Checking to a realistic biological problem: Qualitative Simulation and Model Checking in Genetic Regulatory Networks A presentation of Formal Methods in Biology Justin Hogg email@example.com
Milestones of bacterial genetic research: 1944 Avery's pneumococcal transformation experiment shows that DNA is the hereditary material 1946 Lederberg & Tatum describes bacterial conjugation using biochemical
BENG 221 Mathematical Methods in Bioengineering Project Report A Mathematical Model of a Synthetically Constructed Genetic Toggle Switch Nick Csicsery & Ricky O Laughlin October 15, 2013 1 TABLE OF CONTENTS
Regulation of Protein Synthesis (6.1) Lecture 6 Regulation of Protein Synthesis at the Translational Level Comparison of EF-Tu-GDP and EF-Tu-GTP conformations EF-Tu-GDP EF-Tu-GTP Next: Comparison of GDP
Purpose To consolidate understanding of protein synthesis. To explain the role of transcription factors and hormones in switching genes on and off. Play the transcription initiation complex game Regulation
Induction of Enzyme Activity in Bacteria:The Lac Operon Preparation for Laboratory: Web Tutorial - Lac Operon - submit questions I. Background: For the last week you explored the functioning of the enzyme
CHAPTER 6: RECOMBINANT DNA TECHNOLOGY YEAR III PHARM.D DR. V. CHITRA INTRODUCTION DNA : DNA is deoxyribose nucleic acid. It is made up of a base consisting of sugar, phosphate and one nitrogen base.the
Metabolism & Enzymes 2007-2008 From food webs to the life of a cell energy energy energy Flow of energy through life Life is built on chemical reactions transforming energy from one form to another organic
Biotechnology and Recombinant DNA (Chapter 9) Lecture Materials for Amy Warenda Czura, Ph.D. Suffolk County Community College Primary Source for figures and content: Eastern Campus Tortora, G.J. Microbiology
Ch 4: Energy and Cellular Metabolism Energy as it relates to Biology Chemical reactions Enzymes and how they speed rxs Metabolism and metabolic pathways Catabolism (ATP production) Anabolism (Synthesis
Biotechnology Chapter 20: Biotechnology: DNA Technology & Genomics The BIG Questions How can we use our knowledge of DNA to: o Diagnose disease or defect? o Cure disease or defect? o Change/improve organisms?
What is the difference between basal and activated transcription? Regulation of Transcription I. Basal vs. activated transcription for mrna genes A. General transcription factor (TF) vs. promoterspecific
13.4 Gene Regulation and Expression Lesson Objectives Describe gene regulation in prokaryotes. Explain how most eukaryotic genes are regulated. Relate gene regulation to development in multicellular organisms.
Viruses Chapter 10: Viruses Lecture Exam #3 Wednesday, November 22 nd (This lecture WILL be on Exam #3) Dr. Amy Rogers Office Hours: MW 9-10 AM Too small to see with a light microscope Visible with electron
Transcription: RNA Synthesis, Processing & Modification 1 Central dogma DNA RNA Protein Reverse transcription 2 Transcription The process of making RNA from DNA Produces all type of RNA mrna, trna, rrna,
C H A P T E R E I G H T 8 C o n t r o l o f G e n e E x p r e s s i o n An organism's DNA encodes all of the RNA and protein molecules that are needed to make its cells. Yet a complete description of the
(6) 1. Compare and contrast operon vs gene fusions: (a) Draw a simple diagram showing each type of fusion with transcription and translation start and stop sites the mrna transcript expected the expected
Health and Life Sciences Faculty Course Title: Biological and Forensic Science Module code: 216 BMS Module Title: Molecular Genetics Overview of the Recombinant DNA technology- the process of subcloning
Chapter 8: An Introduction to Metabolism Name Period Concept 8.1 An organism s metabolism transforms matter and energy, subject to the laws of thermodynamics 1. Define metabolism. The totality of an organism
Expression and Purification of Recombinant Protein in bacteria and Yeast Presented By: Puspa pandey, Mohit sachdeva & Ming yu DNA Vectors Molecular carriers which carry fragments of DNA into host cell.
Enzymes Spontaneous Reactions May occur quickly or slowly Enzymes speed up chemical reactions!! (But how, Ms. Robinson????) An enzyme is a macromolecule that acts as a catalyst a chemical agent that speeds
BIOTECHNOLOGY What can we do with DNA? Biotechnology Manipulation of biological organisms or their components for research and industrial purpose Usually manipulate DNA itself How to study individual gene?
Viruses and Prokaryotes Cellular Basis of Life Q: Are all microbes that make us sick made of living cells? 20.1 What is a virus? WHAT I KNOW SAMPLE ANSWER: A virus is a tiny particle that can make people
Chapter 6: Biological Networks 6.4 Engineering Synthetic Networks Prof. Yechiam Yemini (YY) Computer Science Department Columbia University Overview Constructing regulatory gates A genetic toggle switch;
DNA Fingerprinting Unless they are identical twins, individuals have unique DNA DNA fingerprinting The name used for the unambiguous identifying technique that takes advantage of differences in DNA sequence
PYF12 3/21/05 8:04 PM Page 191 Chapter 12 Gene expression and regulation Bacterial genomes usually contain several thousand different genes. Some of the gene products are required by the cell under all
30. Genetics and recombination in bacteria Lecture Outline 11/16/05 Replication in bacteria Types of recombination in bacteria Transduction by phage Conjugation ( mating ) F+ plasmids Hfr s Transformation
Single celled organisms Single Celled Organisms Some organisms, unlike plants and animals, consist of only one cell. These organisms are said to be unicellular An amoeba is another name given to an organism
Energy & Enzymes Life requires energy for maintenance of order, growth, and reproduction. The energy living things use is chemical energy. 1 Energy exists in two forms - potential and kinetic. Potential
Effects of Antibiotics on Bacterial Growth and Protein Synthesis: Student Laboratory Manual I. Purpose...1 II. Introduction...1 III. Inhibition of Bacterial Growth Protocol...2 IV. Inhibition of in vitro
Chapter 3 Protein Structure and Function Broad functional classes So Proteins have structure and function... Fine! -Why do we care to know more???? Understanding functional architechture gives us POWER
Chapter 9 Biotechnology and Recombinant DNA Biotechnology and Recombinant DNA Q&A Interferons are species specific, so that interferons to be used in humans must be produced in human cells. Can you think
RNA & Protein Synthesis Genes send messages to cellular machinery RNA Plays a major role in process Process has three phases (Genetic) Transcription (Genetic) Translation Protein Synthesis RNA Synthesis
DNA and Genetics 1. Which of the following correctly organizes genetic material from the broadest category to the most specific category? A. genome chromosome gene DNA molecule B. genome chromosome DNA
Genetics 301 Sample Final Examination Spring 2003 50 Multiple Choice Questions-(Choose the best answer) 1. A cross between two true breeding lines one with dark blue flowers and one with bright white flowers
BCOR101 Midterm II Wednesday, October 26, 2005 Name Key Please show all of your work. 1. A donor strain is trp+, pro+, met+ and a recipient strain is trp-, pro-, met-. The donor strain is infected with
1 Regulation of enzyme activity Regulation of enzyme activity is important to coordinate the different metabolic processes. It is also important for homeostasis i.e. to maintain the internal environment
Recombinant DNA Technology Dates in the Development of Gene Cloning: 1965 - plasmids 1967 - ligase 1970 - restriction endonucleases 1972 - first experiments in gene splicing 1974 - worldwide moratorium
Chapter 5 Organization and Expression of Immunoglobulin Genes 3 4 5 6 Genetic Models How to account for : ) Vast diversity of antibody specificities ) Presence of Variable regions at the amino end of Heavy
Intro to Metabolism Campbell Chapter 8 http://ag.ansc.purdue.edu/sheep/ansc442/semprojs/2003/spiderlamb/eatsheep.gif http://www.gifs.net Section 8.1 An organism s metabolism transforms matter and energy,
Genetics Lecture Notes 7.03 2005 Lectures 1 2 Lecture 1 We will begin this course with the question: What is a gene? This question will take us four lectures to answer because there are actually several
Biol 205 Exam 1 TEST FORM A Spring 2008 NAME Fill out both sides of the Scantron Sheet. On Side 2 be sure to indicate that you have TEST FORM A The answers to Part I should be placed on the SCANTRON SHEET.
Introduction to Enzyme Kinetics: Assay of β-galactosidase Page 1: Introduction Enzymes are biological molecules that function as catalysts to facilitate specific chemical reactions. Any chemical reaction
Catalysis by Enzymes Enzyme A protein that acts as a catalyst for a biochemical reaction. Enzymatic Reaction Specificity Enzyme Cofactors Many enzymes are conjugated proteins that require nonprotein portions
7. 2 Regulation of gene expression We all start off as one stem cell (a fertilised ovum) that divides to give a ball of cells. These cells then differentiate and become specialised to carry out specific
Biotechnology and Recombinant DNA Recombinant DNA procedures - an overview Biotechnology: The use of microorganisms, cells, or cell components to make a product. Foods, antibiotics, vitamins, enzymes Recombinant
Using chromosomal laci Q1 to control expression of genes on high copy number plasmids in Escherichia coli Christopher B Glascock Michael J Christopher B. Glascock, Michael J. Weickert; Gene 223; 1998 :
Chapter 8 An Introduction to Metabolism PowerPoint Lecture Presentations for Biology Eighth Edition Neil Campbell and Jane Reece Lectures by Chris Romero, updated by Erin Barley with contributions from
Common Course Topics Biology 1406: Cell and Molecular Biology 1. Introduction to biology --the scientific study of organisms --properties of life --assumptions, methods and limitations of science --underlying