Chapter 9

You will not be responsible for all material in this chapter. Only the material concerning operons and whatever is pointed out during lecture.
Regulation of Gene Expression
Chapter 9
I. Overview of Regulation
 9.1 Major Modes of Regulation

 Gene expression: transcription of gene into mRNA followed by translation of mRNA into a protein
 Most proteins are enzymes that carry out biochemical reactions essential for cell growth
 Constitutive proteins are needed at the same level all the time
 Microbial genomes encode many more proteins than are present at any one time
 Regulation is important in all cells and helps conserve energy and resources
 Two major levels of regulation in the cell
 One controls the activity of preexisting enzymes
 Posttranslational regulation
 Very rapid process (seconds)
 One controls the amount of an enzyme
 Regulate level of transcription
 Regulate translation
 Slower process (minutes)

An Overview of Mechanisms of Regulation
II. DNA-Binding Proteins and Regulation of Transcription

9.2 DNA-Binding Proteins
 Recall that mRNA transcripts generally have a
short half-life
 Prevents the production of unneeded proteins
 Regulation of transcription typically requires proteins that can bind to DNA
 Small molecules influence the binding of regulatory proteins to DNA
 Proteins actually regulate transcription
 Most DNA-binding proteins interact with DNA in a sequence-specific manner
 Specificity provided by interactions between amino acid side chains and chemical groups on the bases and
sugar-phosphate backbone of DNA
 Major groove of DNA is the main site of protein binding
 Inverted repeats frequently are binding site for regulatory proteins
 Homodimeric proteins: proteins composed of two identical polypeptides
 Protein dimers interact with inverted repeats on DNA
 Each of the polypeptides binds to one inverted repeat

 Several classes of protein domains are critical for proper binding of proteins to DNA
 Helix-turn-helix
 First helix is the recognition helix
 Second helix is the stabilizing helix
 Many different DNA-binding proteins from Bacteria contain helix-turn-helix
 lac and trp repressors of E. coli

The Helix-Turn-Helix Structure of Some DNA-Binding Proteins

 Classes of Protein Domains
 Zinc finger
 Protein structure that binds a zinc ion
 Typically two or three zinc fingers on proteins that use them for DNA binding
 Leucine zipper
 Leucine residues are spaced every seven amino acids
 Does not interact directly with DNA

Models of Protein Substructures in DNA-Binding Proteins
 Multiple outcomes after DNA binding are possible
1) DNA-binding protein may catalyze a specific reaction on the DNA molecule (i.e., transcription by RNA polymerase)
2) The binding event can block transcription (negative regulation)
3) The binding event can activate transcription (positive regulation)

9.3 Negative Control of Transcription: Repression and Induction

 Several mechanisms for controlling gene expression in bacteria
 These systems are greatly influenced by environment in which the organism is growing
 Presence or absence of specific small molecules
 Interaction between small molecules and DNA-binding proteins result in control of transcription or translation
 Negative control: a regulatory mechanism that stops transcription
 Repression: preventing the synthesis of an enzyme in response to a signal
 Enzymes affected by any repression make up a small fraction of total proteins in the cell
 Typically affects anabolic enzymes (i.e., arginine biosynthesis)

Enzyme Repression
Operons Repression
 Negative Control (cont’d)
 Induction: production of an enzyme in response to a signal
 Typically affects catabolic enzymes (i.e., lac operon)
 Enzymes are synthesized only when they are needed
 no wasted energy

Enzyme Induction
Operons Induction
 Inducer: substance that induces enzyme synthesis
 Corepressor: substance that represses enzyme synthesis
 Effectors: collective term for inducers and repressors
 Effectors affect transcription indirectly by binding to specific DNA-binding proteins
 Repressor molecules bind to an allosteric repressor protein
 Allosteric repressor becomes active and binds to region of DNA near promoter called the operator
 Operon: cluster of genes arranged in a linear fashion whose expression is under control of a single operator
 Operator is located downstream of the promoter
 Transcription is physically blocked when repressor binds to operator
 Enzyme induction can also be controlled by a repressor
 Addition of inducer inactivates repressor and transcription can proceed
 Repressor’s role is inhibitory so it is called negative control

The Process of Enzyme Repression
The Process of Enzyme Induction

9.4 Positive Control of Transcription

 Positive control: regulator protein activates the binding of RNA polymerase to DNA
 Maltose catabolism in E. coli
 Maltose activator protein cannot bind to DNA unless it first binds maltose
 Activator proteins bind specifically to certain DNA sequence
 Called activator binding site, not operator
 Promoters of positively controlled operons only weakly bind RNA polymerase
 Activator protein helps RNA polymerase recognize promoter
 May cause a change in DNA structure
 May interact directly with RNA polymerase
 Activator-binding site may be close to the promoter or several hundred base pairs away
Computer Model of Positive Regulatory Protein and DNA
Activator Protein Interactions with RNA Polymerase
 Genes for maltose are spread out over the chromosome in several operons
 Each operon has an activator-binding site
 Multiple operons controlled by the same regulatory protein are called a regulon
 Regulons also exist for negatively controlled systems

III. Sensing and Signal Transduction

9.5 Two-Component Regulatory Systems

 Prokaryotes regulate cellular metabolism in response to environmental fluctuations
 External signal is not always transmitted directly to the target to be regulated
 Signal transduction: External signal can be detected by a sensor and transmitted to regulatory machinery
 Most signal transduction systems are two-component regulatory systems
 Made up of two different proteins
 Sensor kinase: (cytoplasmic membrane) detects environmental signal and autophosphorylates
 Response regulator: (cytoplasm) DNA-binding protein that regulates transcription
 Absent in bacteria that live as parasites of higher organisms
 Almost 50 different two-component systems in E. coli
 Examples include phosphate assimilation, nitrogen metabolism, and osmotic pressure response
 Some signal transduction systems have multiple regulatory elements
 Some Archaea also have two-component regulatory systems

Control of Gene Expression by a Two-Component System

Examples of Two-Component Regulatory Systems

9.6 Quorum Sensing

 Prokaryotes can respond to the presence of other cells of the same species
 Quorum sensing: mechanism by which bacteria assess their population density
 Ensures sufficient number of cells are present before initiating a response that requires a certain cell density to have an effect (i.e., toxin production in pathogenic bacterium)
 Each species of bacterium produces a specific autoinducer molecule
 Diffuses freely across the cell envelope
 Reaches high concentrations inside cell only if many cells are near
 Binds to specific activator protein and triggers transcription of specific genes
 Several different classes of autoinducers
 Acyl homoserine lactone was the first autoinducer to be identified
 Quorum sensing first discovered as mechanism regulating light production in bacteria including V. fischeri
 Lux operon encodes bioluminescence

Quorum Sensing
Bioluminescent Bacteria Producing the Enzyme Luciferase
 Examples of Quorum Sensing
 P. aeruginosa switches from free living to growing as a biofilm
 Virulence factors of S. aureus
 Quorum sensing is present in some microbial eukaryotes
 Quorum sensing likely exists in Archaea

9.7 Regulation of Chemotaxis

 Modified two-component system used in chemotaxis to
 Sense temporal changes in attractants or repellents
 Regulate flagellar rotation
 Three main steps
1) Response to signal
2) Controlling flagellar rotation
3) Adaptation
 Step1: Response to Signal
 Sensory proteins in cytoplasmic membrane sense presence of attractants and repellents
 Methyl-accepting chemotaxis proteins (MCPs)
 MCPs bind attractant or repellent and initiate interactions that eventually affect flagellar rotation
 Step 2: Controlling Flagellar Rotation
 Controlled by CheY protein
 CheY results in counterclockwise rotation and runs
 CheY-P results in clockwise rotation and tumbling
 Step 3: Adaptation
 Feedback loop
 Allows the system to reset itself to continue to sense the presence of a signal
 Involves modification of MCPs

Interactions of MCPs, Che Proteins and the Flagellar Motor
9.8 Control of Transcription in Archaea

 Archaea use DNA-binding proteins to control transcription
 More closely resembles control by Bacteria than Eukarya

Repression of Genes for Nitrogen Metabolism in Archaea
IV. Global Regulatory Mechanisms

9.9 Global Control and the lac Operon

 Global control systems: regulate expression of many different genes simultaneously
 Catabolite repression is an example of global control
 Synthesis of unrelated catabolic enzymes is repressed if glucose is present in growth medium
 lac operon is under control of catabolite repression
 Ensures the “best” carbon and energy source is used first
 Diauxic growth: two exponential growth phases
Diauxic Growth of E. coli on Glucose and Lactose
 Dozens of catabolic operons affected by catabolite repression
 Enzymes for degrading lactose, maltose, and other common carbon sources
 Flagellar genes are also controlled by catabolite repression
 No need to swim in search of nutrients

9.10 The Stringent Response
 In natural environments nutrients appear/disappear rapidly
 Stringent response: global control mechanism triggered by amino acid starvation
 Triggered by (p)ppGpp
 Alarmones: produced by RelA to signal amino acid starvation
 Stringent response only in Bacteria
 Achieves balance within the call between protein production and protein requirements

9.11 Other Global Control Networks

 Several other global control systems in E. coli
 Heat shock response: largely controlled by alternative sigma factors
 Heat shock proteins: counteract damage of denatured proteins and help cell recover from temperature stress
 Very ancient proteins
 Heat shock response also occurs in Archaea
Control of Heat Shock in Escherichia coli
Examples of Global Control Systems Known in E. coli

V. Regulation of Development in Model Bacteria

9.12 Sporulation in Bacillus

 Regulation of development in model bacteria
 Some prokaryotes display the basic principle of differentiation
 Endospore formation in Bacillus
 Form inside mother cell
 Triggered by adverse external conditions (i.e., starvation or dessication)

Control of Endospore Formation in Bacillus
9.13 Caulobacter Differentiation

 Caulobacter provide another example of differentiation
 Two forms of cells
 Swarmer cells: dispersal role
 Stalked cells: reproductive role
 Many details are still uncertain
 External stimuli and internal factors do play a role in affecting life cycle

Cell Cycle Regulation in Caulobacter
VI. RNA-Based Regulation

9.14 RNA Regulation and Antisense RNA
 Regulatory RNA molecules exert their effects by base pairing with mRNA
 Double-stranded region prevents translation of mRNA
 These small RNAs (~100 nucleotides) are called antisense RNAs
 Each antisense RNA can regulate multiple mRNAs
 Transcription of antisense RNA is enhanced when its target genes need to be turned off
 Some antisense RNA actually enhance translation

9.15 Riboswitches

 Riboswitches: RNA domains in an mRNA molecule that can bind small molecules to control translation of mRNA
 Located at 5′ end of mRNA
 Binding results from folding of RNA into a 3-D structure
 Similar to a protein recognizing a substrate
 Riboswitch control is analogous to negative control
 Found in some bacteria, fungi, and plants
Regulation by Riboswitch

9.16 Attenuation

 Transcriptional control that functions by premature termination of mRNA synthesis
 Control exerted after the initiation of transcription, but before its completion
 First example was the tryptophan operon in E. coli
 mRNA stem-loop structure and synthesis of leader peptide are determining factors in attenuation
 Genomic evidence suggests attenuation exists in Archaea

Attenuation and the Leader Peptide
Mechanism of Attenuation