The Microbial World

BIO20002/BIO60002

Dr Vito Butardo
AS211 Applied Sciences Building
Phone 9214 3715
E-mail vbutardo@swin.edu.au
Copyright notice
Topic 4
Microbial Genetics
Dr Vito Butardo Jr
AS211 Applied Sciences Building
Chapter 7

Microbial Regulatory
Systems Copyright © 2022 Pearson Education Ltd. All Rights
Reserved.
Learning Objectives:

1. Describe the role of DNA binding

proteins in transcriptional regulation
2. Understand the global control of
microbial regulatory systems
3. Identify the mechanism of regulation for
enzymes and other proteins

Copyright © 2022 Pearson Education Ltd. All Rights Reserved.

I. DNA-Binding Proteins and Transcriptional
I

Regulation

7.1 DNA-Binding Proteins

7.2 Transcription Factors and Effectors
7.3 Repression and Activation
7.4 Transcription Controls in Archaea
7.1 DNA-Binding Proteins (1 of 5)

Bacterial and archaeal gene arrangement differs from eukaryotes

Lack introns
Can be arranged in operons: two or more genes transcribed under control of promoter region (single regulatory site)
located upstream of where RNA polymerase initiates transcription
Bacterial and archaeal promoters characterized by distinct nucleotide
sequences recognized and bound by DNA-binding proteins
Allows RNA polymerase to bind, transcription to occur
Animation: Overview of operons
7.1 DNA-Binding Proteins (2 of 5)

Interaction of Proteins with Nucleic Acids

Small molecules influence the binding of regulatory proteins to DNA (Figure 7.1)
• turns transcription on/off
Protein–nucleic acid interactions may be site-specific or nonspecific (attaches
anywhere)
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
Figure 7.1 Gene Expression and Regulation of
Protein Activity
7.1 DNA-Binding Proteins (3 of 5)

Interaction of Proteins with Nucleic Acids

Inverted repeats (nucleotide sequence followed downstream by inverted complement) frequently are specific
binding sites for regulatory proteins (Figure 7.2)
DNA-binding proteins often homodimeric (two identical polypeptides)
• Each polypeptide has a domain (region with specific structure and function) that binds to one inverted repeat
Figure 7.2 DNA-Binding Proteins
7.1 DNA-Binding Proteins (4 of 5)

Structure of DNA-Binding Proteins

helix-turn-helix (Figure 7.3a)
• two α-helices connected by a short “turn” sequence
• first helix: recognition helix (interacts specifically with DNA)
• second helix: stabilizing helix (interacts with first helix through hydrophobic interactions)
• Many different DNA-binding proteins from Bacteria contain helix-turn-helix
• lac and trp repressors of E. coli
Figure 7.3 The Helix-Turn-Helix Structure of Some
DNA-Binding Proteins
7.1 DNA-Binding Proteins (5 of 5)

Structure of DNA-Binding Proteins

zinc finger
• frequently found in eukaryotic regulatory proteins
• binds a zinc ion
leucine zipper
• contains regularly spaced leucine residues
• hold two recognition helices in the correct orientation to bind DNA
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7.2 Transcription Factors and Effectors (1 of 2)

More transcription leads to more mRNA for translation and more protein
product
Mechanisms of Transcription Factors
Transcription factors: Proteins that control the rate of transcription by binding to specific DNA
• Activator protein (Figure 7.4a) turns on transcription
• Binds DNA and recruits RNA polymerase or sigma factor to promoter region
• Repressor protein turns off expression
• Binds operator region of DNA downstream of promoter
Figure 7.4 Transcription Factors, Effectors,
and Allosterism
7.2 Transcription Factors and Effectors (2 of 2)

Effectors: Small molecules that control binding of activators and repressors

Typically cell metabolites (e.g., substrates, products) or structural analogs
Allosteric proteins: Conformation altered when effector molecule binds
Since transcription factors are allosteric, conformational change determines whether transcription factor can bind D
NA
Inducers turn on transcription, corepressors turn off transcription
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7.3 Repression and Activation (1 of 8)

Enzyme Repression and Induction

Enzyme repression: preventing the synthesis of an enzyme unless product is absent
from culture medium; excess of product decreases enzyme synthesis (Figure 7.5a)
• Specific effect (synthesis of all other enzymes continues normally)
• Widespread as control for production of amino acid and nucleotide precursors
• Usually, final product of a biosynthetic pathway is the corepressor effector
molecule
• Typically affects biosynthetic/anabolic enzymes
Figure 7.5 Enzyme Repression and
Expression of the Arginine Operon
Operons: Repression
7.3 Repression and Activation (2 of 8)

Enzyme Repression and Induction

Enzyme induction:
• opposite of repression
• production of an enzyme in response to presence of substrate
• typically affects degradative/catabolic enzymes (e.g., lac operon, Figure 7.6)
• ensures enzymes are synthesized only when needed
Figure 7.6 Enzyme Induction and Expression
of the Lactose Operon
7.3 Repression and Activation (3 of 8)

Mechanisms of Repression and Derepression

Repressors turn off transcription
Corepressors only bind DNA in presence of its effector.
Example: Arginine becomes corepressor when plentiful
• Binds arginine repressor (ArgR)
• Results in allosteric change and operator binding
• Since arg mRNA is polycistronic, all peptides encoded are repressed
7.3 Repression and Activation (4 of 8)

Mechanisms of Repression and Derepression

Some repressors bind in absence of effector, e.g., lac operon (derepression)
• LacI (lactose repressor) binds to operator, blocks transcription
• If LacI effector present, combines with repressor, causing allosteric change that
prevents LacI from binding DNA
• Transcription can proceed
• Corepressor = inducer (inactivates repressor)
• Allolactose and isopropylthiogalactosie (IPTG) are lac inducers
7.3 Repression and Activation (5 of 8)

Mechanisms of Repression and Derepression

Repressor’s role is inhibitory (preventing mRNA synthesis), so it is called negative control
Genes are not turned on and off completely; often very low level of basal transcription when “fully repressed”
7.3 Repression and Activation (6 of 8)

Mechanisms of Activation
Some operons transcribed only if activator protein first bound to DNA (Figure 7.7)
Promoter sequences are poor matches to consensus promoter sequences, thus only weakly bind RNA polymerase
Positive control: regulator protein facilitates transcription
Figure 7.7 Activator Protein Interactions With
RNA Polymerase
7.3 Repression and Activation (7 of 8)

Mechanisms of Activation
Activator proteins help RNA polymerase recognize promoter.
• May bend DNA structure (Figure 7.8)
• May interact directly with RNA polymerase
Example: maltose catabolism in E. coli (Figure 7.9)
• Maltose activator protein (MalT) cannot bind to DNA unless it first binds maltose
(effector/inducer)
• Binding of MalT allows RNA polymerase to transcribe
Activator proteins bind specifically to activator-binding site (specific DNA sequence,
not called an operator)
Figure 7.8 Computer Model of a Positive Regulatory
Protein Interacting With DNA
Figure 7.9 Positive Control of Enzyme
Induction in the Maltose Operon
7.3 Repression and Activation (8 of 8)

Operons versus Regulons

Genes for maltose are spread out over the chromosome in several operons. (Figure
7.10)
• Each operon has an activator-binding site
• Maltose activator protein controls transcription of more than one operon
• Multiple operons controlled by the same regulatory protein are called a regulon
(e.g., maltose regulon)
Regulons also exist for negatively controlled systems (e.g., arginine regulon)
Multiple control features common in many operons and regulons
Figure 7.10 Maltose Regulon of Escherichia
coli
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III. Global Control

7.8 The lac Operon

7.9 Stringent and General Stress Responses
7.10 The Phosphate (Pho) Regulon
7.11 The Heat Shock Response
7.8 The lac Operon (1 of 5)

Global control systems: regulate transcription of many different genes in more

than one regulon (Table 7.2)
May include activators, repressors, signal molecules, two-component regulatory systems, regulatory RNA, alternative
sigma factors as components
Table 7.2 Examples of Global Control Systems
Known in Escherichia colia
System Signal Primary activity of regulatory Number of genes
protein regulated
Aerobic respiration Presence of O2 Repressor (ArcA) > 50
Anaerobic respiration Lack of O2 Activator (FNR) > 70
Catabolite repression Cyclic AMP level Activator (CRP) > 300
Heat shock Temperature Alternative sigma factors (RpoH > 36
and RpoE)
Nitrogen utilization NH3 limitation Activator (NRI)/alternative sigma > 12
factor (RpoN)
Oxidative stress Oxidizing agents Activator (OxyR) > 30
SOS response Damaged DNA Repressor (LexA) > 20
General stress response Stress conditions Alternative sigma factor (RpoS) > 400
aFor many of the global control systems, regulation is complex. A single regulatory protein can play more than one role. For
instance, the regulatory protein for aerobic respiration is a repressor for many promoters but an activator for others, whereas the
regulatory protein for anaerobic respiration is an activator protein for many promoters but a repressor for others. Regulation can
also be indirect or require more than one regulatory protein. Many genes are regulated by more than one global system.
7.8 The lac Operon (2 of 5)

Lactose operon and maltose regulon respond to global controls

Catabolite Repression
controls use of carbon sources if more than one present
• glucose always used first
Synthesis of unrelated catabolic enzymes (e.g., lactose operon and maltose regulon)
is repressed if glucose is present in growth medium
also called “glucose effect”
ensures that the “best” carbon and energy source is used first
7.8 The lac Operon (3 of 5)

Catabolite Repression
Can lead to diauxic growth: two exponential growth phases if two energy sources available (Figure 7.21)
• Better energy source consumed first, growth stops
• After lag, growth resumes with second energy source
Figure 7.21 Diauxic Growth of Escherichia coli on a
Mixture of Glucose and Lactose
7.8 The lac Operon (4 of 5)

Cyclic AMP and Cyclic AMP Receptor Protein

In catabolite repression, transcription is controlled by the cyclic AMP receptor protein (CRP), an activator protein, and
is a form of activation
CRP is allosteric and binds to DNA only if it has first bound cyclic adenosine monophosphate (cyclic AMP or cAMP)
(Figure 7.22)
• regulatory nucleotide derived from adenosine
• synthesized by adenylate cyclase
Figure 7.22 Cyclic AMP
7.8 The lac Operon (5 of 5)

Cyclic AMP and Cyclic AMP Receptor Protein

For lac genes to be transcribed (Figure 7.23):
• Cyclic AMP level must be high enough for CRP protein to bind to CRP-binding site (positive control)
• Lactose or another inducer must be present to prevent lactose repressor (LacI) binding (negative control)
Figure 7.23 Overall Regulation of the Lac
System
Animation: Summary of lac operon
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7.10 The Phosphate (Pho) Regulon (1 of 4)

Phosphorus essential for DNA, RNA, membrane synthesis, energy generation,

cell signaling
Inorganic phosphate (PO43− or Pi) often limiting in environment
Phosphate (Pho) regulon: two-component regulatory system that responds to
Pi limitation
7.10 The Phosphate (Pho) Regulon (2 of 4)

Streptomyces and Phosphate

Streptomyces: gram-positive genus, many of which produce antibiotics
Antibiotic production is energy-intensive but limited by high phosphate
Low phosphate signals competition for resources, resulting in antibiotic production
7.10 The Phosphate (Pho) Regulon (3 of 4)

Streptomyces and Phosphate

Two-component Pho regulatory system senses Pi limitation
• Membrane-bound histidine kinase sensor protein (PhoR) and cytoplasmic transcription
regulator (PhoP)
• Low environmental Pi triggers PhoR kinase, yielding phosphorylated PhoP (PhoP-P) (Figure
7.26)
• PhoP-P binds conserved promoter regions (“Pho boxes”)
• Signals RNA polymerase to bind, activating transcription for Pi uptake and some genes for
antibiotic production
PhoP-P binds some promoters weakly without recruiting sigma subunit, blocking RNA
polymerase and repressing linked gene (e.g., nitrogen metabolism)
Figure 7.26 The Phosphate (Pho) Regulon of
Streptomyces
7.10 The Phosphate (Pho) Regulon (4 of 4)

Pathogenesis and the Pho Regulon

Pho regulon can control some aspects of pathogenesis
E. coli O157:H7 has Pho regulon of 40+ genes.
• Phosphate limitation leads to increased fitness/survivability
Implicated in regulating biofilm formation, antimicrobial resistance, toxin production in other pathogenic bacteria
(e.g., Vibrio cholerae, Pseudomonas)
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7.11 The Heat Shock Response (1 of 2)

Heat shock response: global control mechanism to protect cells from protein
denaturation resulting from heat, high solvent levels, osmotic stress,
ultraviolet (UV) light
Heat Shock Proteins
Temperature and stress can generate large amounts of inactive proteins that need to be refolded or degraded
Heat shock proteins: counteract damage of denatured proteins and help cell recover from stress
• five major classes: Hsp100 (proteases that degrade denatured/aggregated proteins), Hsp90, Hsp70 (DnaK), Hs
p60 (GroEL), Hsp10 (GroES)
7.11 The Heat Shock Response (2 of 2)

The Alternative Sigma Factor RpoH

Heat shock response controlled by alternative sigma factor RpoH (Figure 7.27)
• Controls heat shock protein expression
• Degraded within 1–2 minutes of synthesis
• When heat shock (or other stressor) occurs, RpoH degradation inhibited, level increases,
increasing transcription of operons whose promoters are recognized
• RpoH degradation depends on level of DnaK, which inactivates RpoH
• RpoH mRNA basepairs with itself, regulating translation.
Also heat shock response in Archaea and eukaryotes
Figure 7.27 Control of Heat Shock in
Escherichia coli
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V. Regulation of Enzymes and Other Proteins

7.15 Feedback Inhibition

7.16 Post-Translational Regulation
7.15 Feedback Inhibition (1 of 2)

Feedback inhibition: mechanism for temporarily turning off the reactions in a

biosynthetic pathway (Figure 7.34a)
End product of the pathway binds to an early (often the first) enzyme in the pathway, thus shutting down the
pathway because no intermediates are generated
Reversible reaction: once levels of end product are limiting, pathway functions
Inhibited enzyme has two binding sites: active (substrate-binding) and allosteric (end product binds)
• Binding at allosteric site changes conformation, preventing substrate binding
Figure 7.34 Inhibition of Enzyme Activity
7.15 Feedback Inhibition (2 of 2)

Some pathways controlled by feedback inhibition use isoenzymes: different

proteins that catalyze the same reaction but are subject to different regulators
example: 3 DAHP synthases for aromatic amino acids are inhibited incrementally (excess of all three required to shut
down pathway)
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7.16 Post-Translational Regulation (1 of 4)

Phosphorylation and methylation are two common mechanisms for post-

translational regulation
Biosynthetic enzymes can be regulated by other covalent modifications
(attachment or removal of small molecule)
common modifiers include adenosine monophosphate (AMP), adenosine diphosphate (ADP), uridine
monophosphate (UMP)
7.16 Post-Translational Regulation (2 of 4)

Regulation of PII Signal Transduction Proteins

PII proteins are widespread family found in Bacteria, Archaea, and plant plastids
Regulate nitrogen metabolism transcription factors, enzymes, membrane transport proteins
Modifications like uridylylation (addition of UMP), adenylylation (addition of AMP), and phosphorylation affect activity
Post-translational: occurs after protein synthesis
Uridylylation and removal by by GlnD affects ammonia assimilation (Figure 7.35)
Figure 7.35 Post-Translational Regulation of PII and
Glutamine Synthetase
7.16 Post-Translational Regulation (3 of 4)

Regulation of PII Signal Transduction Proteins

GlnD senses glutamine
• If too low, signals that NH3 assimilation needed
• When GlnD is not bound to glutamine, it adds a UMP to PII, forming PII-UMP
One target of PII-UMP is glutamine synthetase adenyltransferase
• Stimulates this enzyme to remove adenyl groups from glutamine synthetase (GS),
increasing its activity and increasing NH3 assimilation
• PII stimulates glutamine synthetase adenyltransferase to adenylate GS, which is less active
Overall effect is energy conservation: GS activity uses ATP
7.16 Post-Translational Regulation (4 of 4)

Inactivation of Sigma Factors

Anti-sigma factors can inactivate sigma factors
example: RpoE and anti-sigma factor RseA under membrane stress (Figure 7.36a)
example: σF and anti-sigma factor SpoIIAB during Bacillus endospore formation
Figure 7.36 Anti-Sigma–Sigma Factor
Interactions
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