Chapter 7
Metabolic Regulation
I. Overview of Regulation
• 7.1 Major Modes of Regulation
7.1 Major Modes of Regulation
• Gene expression: transcription of gene into mRNA
followed by translation of mRNA into protein (Figure 7.1)
•
Most proteins are enzymes that carry out biochemical
reactions
•
Constitutive proteins are needed at the same level all the
time
•
Microbial genomes encode many proteins that are not
needed all the time
•
Regulation helps conserve energy and resources
7.1 Major Modes of Regulation
• Two major levels of regulation in the cell:
•
•
One controls the activity of preexisting enzymes
• Post-translational regulation
• Very rapid process (seconds)
One controls the amount of an enzyme
• Regulates level of transcription
• Regulates translation
• Slower process (minutes)
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7.1 Major Modes of Regulation
• Monitoring gene expression
•
Reporter genes encode for an easy-to-detect product
(Figure 7.2)
• Can be fused to other genes
• Can be fused to regulatory elements
• Example: green fluorescent protein (GFP)
II. DNA-Binding Proteins and Transcriptional
Regulation
• 7.2 DNA-Binding Proteins
• 7.3 Negative Control: Repression and Induction
• 7.4 Positive Control: Activation
• 7.5 Global Control and the lac Operon
• 7.6 Transcriptional Controls in Archaea
7.2 DNA-Binding Proteins
• 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
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•
Proteins actually regulate transcription
7.2 DNA-Binding Proteins
• 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
7.2 DNA-Binding 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
(Figure 7.3)
7.2 DNA-Binding Proteins
• Several classes of protein domains are critical for proper
binding of proteins to DNA
• Helix-turn-helix (Figure 7.4)
• First helix is the recognition helix
• Second helix is the stabilizing helix
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• Many different DNA-binding proteins from Bacteria contain
helix-turn-helix
•
lac and trp repressors of E. coli
7.2 DNA-Binding Proteins
• Classes of protein domains
•
•
Zinc finger
• Protein structure that binds a zinc ion
• Eukaryotic regulatory proteins use zinc fingers for DNA binding
Leucine zipper
• Contains regularly spaced leucine residues
• Function is to hold two recognition helices in the correct
orientation
7.2 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)
7.3 Negative Control: Repression and Induction
• Several mechanisms for controlling gene expression in
bacteria
• These systems are greatly influenced by environment in
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•
•
which the organism is growing
Presence or absence of specific small molecules
Interactions between small molecules and DNA-binding
proteins result in control of transcription or translation
7.3 Negative Control: Repression and Induction
• Negative control: a regulatory mechanism that stops
transcription
• Repression: preventing the synthesis of an enzyme in
response to a signal (Figure 7.5)
• Enzymes affected by repression make up a small fraction of
total proteins
• Typically affects anabolic enzymes (e.g., arginine biosynthesis)
7.3 Negative Control: Repression and Induction
• Negative control (cont'd)
•
Induction: production of an enzyme in response to a signal
(Figure 7.6)
• Typically affects catabolic enzymes (e.g., lac operon)
• Enzymes are synthesized only when they are needed
• No wasted energy
7.3 Negative Control: Repression and Induction
• Inducer: substance that induces enzyme synthesis
• Corepressor: substance that represses enzyme
synthesis
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•
•
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
7.3 Negative Control: Repression and Induction
• 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 (Figure 7.7)
•
Enzyme induction can also be controlled by a repressor
• Addition of inducer inactivates repressor, and transcription
can proceed (Figure 7.8)
•
Repressor's role is inhibitory, so it is called negative
control
7.4 Positive Control: Activation
• Positive control: regulator protein activates the binding of
RNA polymerase to DNA (Figure 7.9)
•
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
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•
Called activator-binding site, not operator
7.4 Positive Control: Activation
• 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 be
several hundred base pairs away (Figure 7.11)
7.4 Positive Control: Activation
• Genes for maltose are spread out over the chromosome
in several operons (Figure 7.12)
• 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
7.5 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
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•
•
•
glucose is present in growth medium (Figure 7.13)
lac operon is under control of catabolite repression
Ensures that the "best" carbon and energy source is used
first
Diauxic growth: two exponential growth phases
7.5 Global Control and the lac Operon
• Cyclic AMP and CRP
•
•
•
In catabolite repression, transcription is controlled by an
activator protein and is a form of positive control (Figure
7.15)
Cyclic AMP receptor protein (CRP) is the activator protein
Cyclic AMP is a key molecule in many metabolic control
systems
• Derived from a nucleic acid precursor
• Is a regulatory nucleotide
7.5 Global Control and the lac Operon
• Dozens of catabolic operons are 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
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7.6 Transcription Controls in Archaea
• Archaea use DNA-binding proteins to control
transcription
• More closely resembles control by Bacteria than Eukarya
•
Repressor proteins in Archaea
• NrpR is an example of an archaeal repressor protein from
Methanococcus maripaludis (Figure 7.16)
• Represses genes involved in nitrogen metabolism
III. Sensing and Signal Transduction
• 7.7 Two-Component Regulatory Systems
• 7.8 Regulation of Chemotaxis
• 7.9 Quorum Sensing
• 7.10 Other Global Control Networks
7.7 Two-Component Regulatory Systems
• Prokaryotes regulate cellular metabolism in response to
environmental fluctuations
• External signal is transmitted directly to the target
• External signal is detected by sensor and transmitted to
regulatory machinery (signal transduction)
• Most signal transduction systems are two-component regulatory
systems
7.7 Two-Component Regulatory Systems
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•
Two-component regulatory systems (Figure 7.17)
• Made up of two different proteins:
• Sensor kinase (in cytoplasmic membrane): detects
environmental signal and autophosphorylates
• Response regulator (in cytoplasm): DNA-binding protein that
regulates transcription
•
Also has feedback loop
• Terminates signal
7.7 Two-Component Regulatory Systems
• Almost 50 different two-component systems in E. coli
•
Examples include phosphate assimilation, nitrogen
metabolism, and osmotic pressure response (Figure 7.18)
•
Some signal transduction systems have multiple
regulatory elements
•
Some Archaea also have two-component regulatory
systems
7.8 Regulation of Chemotaxis
• Modified two-component system used in chemotaxis to
•
•
•
Sense temporal changes in attractants or repellents
Regulate flagellar rotation
Three main steps (Figure 7.19)
1. Response to signal
2. Controlling flagellar rotation
3. Adaptation
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7.8 Regulation of Chemotaxis
• Step 1: Response to signal
•
•
•
Sensory proteins in cytoplasmic membrane sense
attractants and repellents
Methyl-accepting chemotaxis proteins (MCPs)
• Bind attractant or repellent and initiate 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
7.8 Regulation of Chemotaxis
• Step 3: Adaptation
•
Feedback loop
• Allows the system to reset itself to continue to sense the
presence of a signal
• Involves modification of MCPs
7.8 Regulation of Chemotaxis
• Other Taxes
•
•
•
Che proteins also play a role in these
Phototaxis: movement toward light
• Light sensor replaces MCPs
Aerotaxis: movement toward oxygen
• Redox protein monitors oxygen level
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7.9 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 that a sufficient number of cells are present before
initiating a response that, to be effective, requires a certain
cell density (e.g., toxin production in pathogenic bacterium)
7.9 Quorum Sensing
• Each species of bacterium produces a specific
autoinducer molecule (Figure 7.20)
• 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
7.9 Quorum Sensing
• Several different classes of autoinducers
•
•
Acyl homoserine lactone (AHL) was the first autoinducer to
be identified
Quorum sensing first discovered as mechanism
regulating light production in bacteria including Aliivibrio
fischeri (Figure 7.21)
• Lux operon encodes bioluminescence
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7.9 Quorum Sensing
• Examples of quorum sensing
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•
•
•
Virulence factors
Switching from free-living to growing as a biofilm
Quorum sensing is present in some microbial eukaryotes
Quorum sensing likely exists in Archaea
7.9 Quorum Sensing
• Virulence factors
•
Escherichia coli O57:H7
• Shiga toxin–producing strain
• Produces AHL AI-3
• Epinephrine plus norepinephrine plus AI-3 bind to sensor
molecules in plasma membrane
•
Activates motility, enterotoxin production, and production of
virulence proteins (Figure 7.22a)
7.9 Quorum Sensing
• Virulence factors (cont'd)
•
Staphylococcus aureus
• Secretes small peptides that damage host cells or alter host's
immune system
• Under control of autoinducing peptide (AIP)
• Activates several proteins that lead to production of virulence
proteins (Figure 7.22b)
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7.9 Quorum Sensing
• Biofilm formation
•
Pseudomonas aeruginosa
• Produces polysaccharides that increase pathogenicity and
antibiotic resistance
• Two quorum-sensing systems
• Produces AHLs and cyclic di-guanosine monophosphate
(c-di-GMP)
•
Leads to exopolysaccharide production and flagella synthesis
(Figure 7.23)
7.10 Other Global Control Networks
• Several other global control systems
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•
•
•
•
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Aerobic and anaerobic respiration
Catabolite repression
Nitrogen utilization
Oxidative stress
SOS response
Heat shock response
7.10 Other Global Control Networks
• Heat shock response:
•
•
Largely controlled by alternative sigma factors (Figure 7.24)
Heat shock proteins: counteract damage of denatured
proteins and help cell recover from temperature stress
• Very ancient proteins
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•
Heat shock response also occurs in Archaea
IV. Regulation of Development in Model Bacteria
• 7.11 Sporulation in Bacillus
• 7.12 Caulobacter Differentiation
• 7.13 Nitrogen Fixation, Nitrogenase, and Heterocyst
Formation
7.11 Sporulation in Bacillus
• Regulation of development in model bacteria
•
•
Some prokaryotes display the basic principle of
differentiation
Endospore formation in Bacillus (Figure 7.25)
• Controlled by four sigma factors
• Forms inside mother cell
• Triggered by adverse external conditions (i.e., starvation or
desiccation)
7.12 Caulobacter Differentiation
• Caulobacter provides another example of differentiation
• Two forms of cells:
•
•
•
Swarmer cells: dispersal role
Stalked cells: reproductive role
Controlled by three major regulatory proteins (Figure
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7.26)
• External stimuli and internal factors play a role in affecting
life cycle
7.13 Nitrogen Fixation, Nitrogenase, and
Heterocyst Formation
• Nitrogen fixation is process of reducing N2 to NH3
• Only certain prokaryotes can fix nitrogen
• Reaction is catalyzed by nitrogenase
•
•
Composed of dinitrogenase and dinitrogenase reductase
Sensitive to the presence of oxygen
7.13 Nitrogen Fixation, Nitrogenase, and
Heterocyst Formation
• Highly regulated process because it is such an energydemanding process
•
Nif regulon coordinates regulation of genes essential to
nitrogen fixation (Figure 7.27)
•
Oxygen and ammonia are the two main regulatory
effectors
7.13 Nitrogen Fixation, Nitrogenase, and
Heterocyst Formation
• Heterocyst formation (Figure 7.28)
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•
Requires metabolic and morphological changes
• Formation of thickened envelope
• Inactivation of photosystem II
• Expression of nitrogenase
• Patterning of heterocyst differentiation
V. RNA-Based Regulation
• 7.14 Regulatory RNAs: Small RNAs and Antisense RNA
• 7.15 Riboswitches
• 7.16 Attenuation
7.14 Regulatory RNA: Small RNAs and
Antisense RNA
• Regulatory RNA molecules exert their effects by base
pairing with mRNA (Figure 7.29)
• Block a ribosome-binding site (RBS)
• Open up a blocked RBS
• Increase degradation of mRNA
• Decrease degradation of mRNA
7.14 Regulatory RNA: Small RNAs and
Antisense RNA
• Types of small RNAs
•
Small RNAs made by transcribing nontemplate strand of
gene are called antisense RNAs
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• Each antisense RNA can regulate multiple mRNAs
• Transcription of antisense RNA is enhanced when its target
genes need to be turned off
• Some antisense RNAs actually enhance translation
7.14 Regulatory RNA: Small RNAs and
Antisense RNA
• Types of small RNAs (cont'd)
•
•
Trans-sRNAs are encoded in the intergenic region
Binding of trans-sRNA to targets depends on a small protein
• Small proteins called RNA chaperones
• Example of RNA chaperone: Hfq
• Hfq binds to both RNA molecules and to ribonuclease E
(Figure 7.30)
• Helps small molecules maintain correct structure
7.15 Riboswitches
• Riboswitches: RNA domains in an mRNA molecule that
can bind small molecules to control translation of mRNA
(Figure 7.31)
• 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
7.16 Attenuation
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•
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 (Figure
7.32)
mRNA stem–loop structure and synthesis of leader peptide
are determining factors in attenuation (Figure 7.33)
Genomic evidence suggests attenuation exists in
Archaea
VI. Regulation of Enzymes and Other Proteins
• 7.17 Feedback Inhibition
• 7.18 Post-Translational Regulation
7.17 Feedback Inhibition
• Feedback inhibition: mechanism for turning off the
reactions in a biosynthetic pathway (Figure 7.34a)
• End product of the pathway binds to the first enzyme in the
•
•
pathway, thus inhibiting its activity
Inhibited enzyme is an allosteric enzyme (Figure 7.34b)
• Two binding sites: active and allosteric
Reversible reaction
7.17 Feedback Inhibition
• Some pathways controlled by feedback inhibition use
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isoenzymes
•
Isoenzymes
• Different enzymes that catalyze the same reaction but are
subject to different regulatory controls (Figure 7.34c)
7.18 Post-Translational Modification
• Biosynthetic enzymes can also be regulated by covalent
modifications
• Regulation involves a small molecule attached to or
•
•
removed from the protein (Figure 7.35)
Results in conformational change that inhibits activity
Common modifiers include adenosine monophosphate
(AMP), adenosine diphosphate (ADP), inorganic phosphate
(PO42-), and methyl groups (CH3)
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