Bacteriology

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Bacteriology course
Jeong Nam Kim
Department of Microbiology
Pusan National University
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
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)
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)
Figure 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
– Proteins actually regulate transcription
DNA-Binding Proteins
• Most DNA-binding proteins interact with DNA in a sequencespecific 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
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)
Domain containing protein–protein
contacts, holding protein dimer together
DNA-binding domain fitsin
major grooves and along
sugar–phosphate backbone
Inverted repeats
5′ TG T G T G G A A T TGT GA GC GGA T A A C A A T T T C A C A C A 3′
3′ AC AC A C C T T AACA C T C GC C T A T T GT TA AA G T G T G T 5′
Inverted repeats
Figure 7.3
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
• Many different DNA-binding proteins from Bacteria
contain helix-turn-helix
– lac and trp repressors of E. coli
Turn
Recognition
helix
Stabilizing
helix
DNA
Subunits of
binding
protein
Figure 7.4
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
DNA-Binding Proteins
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)
Negative Control: 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
– Interactions between small molecules and DNAbinding proteins result in control of transcription
or translation
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)
Repression
Cell number
Total protein
Arginine added
Arginine
biosynthesis
enzymes
Figure 7.5
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
Induction
Total protein
Cell number
β-Galactosidase
Lactose added
Figure 7.6
Negative Control: Repression and
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
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
arg Promoter arg Operator
argC
argB
RNA
polymerase
argH
Transcription proceeds
Repressor
arg Promoter arg Operator
RNA
polymerase
argC
argB
argH
Corepressor
Transcription blocked
(arginine)
Repressor
Figure 7.7
lac Promoter lac Operator
RNA
polymerase
lacZ
lacY
lacA
Transcription blocked
Repressor
lac Promoter lac Operator
RNA
polymerase
lacZ
lacY
lacA
Transcription proceeds
Repressor
Inducer
(allolactose)
Figure 7.8
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
– Called activator-binding site, not operator
Activatorbinding site
mal Promoter
malE
malF
malG
No transcription
RNA
polymerase
Maltose activator protein
Activatorbinding site
mal Promoter
malE
RNA
polymerase
malF
malG
Transcription proceeds
Maltose activator protein
Inducer
(maltose)
Figure 7.9
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)
Activatorbinding site
Promoter
RNA
polymerase
Transcription
proceeds
Activator protein
Promoter
RNA
polymerase
Activator protein
Transcription
proceeds
Activatorbinding site
Figure 7.11
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