Regulating Gene Expression
• Microbes respond to changing environment
– Alter growth rate
– Alter proteins produced
• Must sense their environment
– Receptors on cell surface
• Must transmit information to chromosome
• Alter gene expression
– Change transcription rate
– Change translation rate
Sensing the Environment
• Two‐component phosopho‐relay signal transduction
– Receptor/Sensor Histidine‐kinase protein in plasma membrane
• Binds to a signal cue
• Activates itself via phosphorylation
p p y
– Cytoplasmic response regulator
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„
Takes phosphate from sensor
Binds chromosome
Alters transcription rate of
multiple genes
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Operonic regulation • Coding vs regulatory sequences
• Regulatory sequences: promoters, operator and activator sequences
• Regulatory proteins: repressors, activators
– Repressors bind operator sequences, block transcription
• Induction vs Derepression
– Activator proteins bind sequences near by promoters, facilitate RNA Pol binding, upregulate transcription The E. coli
The E. coli lac Operon
• Lactose (milk sugar) is used for food
– Cannot pass through plasma membrane
• Lactose permease allows entry
• PMF used to bring lactose inside cell
– Must be converted to glucose to be digested
„
β-galactosidase
β
galactosidase
converts lactose to
glucose and
galactose
People also make βgalactosidase
If not, person is lactoseintolerant
The E. coli
The E. coli lac Operon
• The lacZ gene encodes β‐galactosidase
• The lacY gene encodes lactose permease
– Need both proteins to digest lactose
• Operon
– Multiple genes transcribed from one promoter
Multiple genes transcribed from one promoter
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The E. coli
The E. coli lac Operon
• Repressor protein LacI blocks transcription
– Repressor binds to operator
– Blocks σ factor from binding promoter
gp
• Repressor responds to presence of lactose
– Binds inducer (allolactose) or DNA, not both
– Add lactose → repressor falls off operator
Allolactose cause operon induction
Activation of the lac
Activation of the lac Operon by by cAMP
cAMP‐‐CRP
• Maximum expression requires cAMP and cAMP receptor protein (CRP)
‐ The cAMP‐CRP complex binds to the promoter at ‐60 bp
‐ Interacts with RNA pol, increase rate of transcription initiation
• CRP acts as activator only when bound to cAMP
Catabolite Repression
• Glucose is present, lac operon is OFF (no transcription)
• Two mechanisms involved
1. High glucose → low cAMP levels → CRP inactive
– Can’t bind operon → low level of lac transcription
•
•
•
•
•
PTS dependent glucose
uptake
P-transfer from IIA-P to IIB for
Glucose uptake
IIA becomes available
IIA: inhibitor of AC
cAMP level reduced
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Catabolite Repression
2. Inducer exclusion:
– High glucose → high IIA
– IIA inhibits LacY permease
– Reducing intracellular lactose
• Importance of catabolite repression
– Sequential sugar catabolism
– diauxic growth
Animation: The E. coli lac Operon
Arabinose operon
• Regulation by dual role regulatory protein AraC
• “AraC” acts as repressor to block transcription (no arabinose)
• Acts also as activator when bound to “arabinose” (the inducer)
– Operators O1, O2 and araI control AraC and AraBAD proteins expression
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Ara Operon Controls
• Senario I: No arabinose present
– “AraC” forms long dimeric conformation, blocks transcription (binding O2, araI1)
• Senario II: arabinose added
– changes AraC dimeric conformation
• acts as activator
• Stimulates binding of RNA polymerase
+ arabinose
Trp operon
operon: Repression and Attenuation
: Repression and Attenuation
• trp operon
– Cell must make the amino acid tryptophan
• Trp operon codes and regulates biosythetic enzymes
• When tryptophan is plentiful, cell stops synthesis
• Regulation by two mechanisms
1. Repression: Trp repressor must bind tryptophan to bind DNA
• Opposite of lac repressor
Repressor + Tryptophan
Transcription repressed
Transcriptional Attenuation of the trp Operon
2. Attenuation: a regulatory mechanism in which translation of a leader peptide affects transcription of a downstream structural gene
The attenuator region
of the trp operon
has 2 trp codons
and is capable of
forming stem-loop
structures.
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Transcriptional Attenuation Mechanism of the trp Operon
High tryptophan
Low tryptophan
Animation: Transcriptional Attenuation
Animation: Sigma Factor Regulation
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•
•
•
•
σ factors regulate transcription of all genes
Recognize promoter sequences differentially
Specific to a subset of genes
Direct RNA Pol to start transcription
Alternative σ factors used for global transcriptional regulation of related genes
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How are sigma factors regulated?
• Temperature‐sensitive mRNA 2°‐structure
– at 42°C σ70 degraded rapidly
– Allows translation of σ32 (σΗ) only at high temperature
• Synthesis of proteins that inhibit σ factors
– Anti‐σ factors block σ activity until needed
– Anti‐anti‐σ factors respond to environment
Small Regulatory RNAs
• found in bacterial intergenic regions
• Regulate transcription or interfere with translation
• antisense nature of sRNA allows its binding to mRNA
‐ ex. pairing of RNAIII with 5’‐end of mRNA prevents ribosome assembly, ie. halting translation
– mRNA degradation
RNA d
d ti
by dsRNA specific Read more: RNAIII represses virulence factors
enzyme RNaseIII
– RNAIII a multi‐locus
global regulator of several vir. factors
• Protein A (spa)
• Coaggulase (Sa1000) • CRISPR system: Clustered Regularly Interspersed Short Palindromic Repeats
• CRISPR arrays
– Repeats: tandem array palindromes (4 to >100), 28‐40 bases, form stem‐loop in transcripts, 3’ terminus GAA(AC/G)
– Spacers: 25‐40 bp, identical to phage or plasmid and some chromosomal seq., mostly sense, some antisense
• Leader: ~550 bp, immediately 5’ to array , AT rich, promoter
• CAS: clustered gene families, only present in genomes containing CRISPRs, code for RNA or DNA active proteins, possibly involved in processing arrays and psiRNA
1. CRISPERS: Nature Reviews (Microbiology), 6:181, 2008
2. Evolution of CRISPRS CAS prteins , Nature Reviews(Microbiology), 9:467, 2011
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CRISPR: a global response to foreign DNA or RNA
• specificity sensitive to single nucleotide difference
• maintains memory
• a primitive nucleic acid based adaptive immunity?
Quorum Sensing
• Cells work together coordinately at high cell density
– V. fischeri becomes bioluminescent
– Many bacteria form biofilms
• Discovered in Vibrio fischeri, a bioluminescent bacterium that colonizes the light organ of the Hawaiian squid
Quorum Sensing: Bioluminescence induction
• Induction of a quorum‐sensing genes need accumulation of a secreted small molecule called an autoinducer
– Homoserine lactone for V. fishcheri
• At
At a certain extracellular concentration, the secreted a certain extracellular concentration the secreted
autoinducer reenters cells
‐ binds to a regulatory molecule, which in the case of Vibrio fischeri is LuxR
‐ The LuxR‐autoinducer complex then activates transcription of the luciferase target genes that confer bioluminescence
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Quorum Sensing: Bioluminescence induction
Animation: Quorum Sensing
Animation: Summary
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Regulatory proteins help the cell sense and react to changes in its internal environment.
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Two‐component signal transduction systems help the cell sense its external environment.
The lacZYA
The
lacZYA operon is regulated as follows:
operon is regulated as follows:
‐ Operon is off when LacI binds to the operator. ‐ Operon is on when allolactose binds to LacI; cAMP‐
CRP are bound to the promoter (and there is no glucose around). ● The tryptophan operon is regulated by repression and attenuation (premature transcript termination).
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Summary
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Sigma factors are controlled by alternate transcription and translation, proteolysis, and anti‐sigma factors ●
Small regulatory RNAs can bind to mRNA and inhibit translation and cause mRNA degrade. CRISPR is a global reaction against invading foreign RNA or DNA
reaction against invading foreign RNA or DNA
Bacterial genes are regulated by a hierarchy of regulators that form integrated gene circuits.
‐ Example: Chemotaxis
● In quorum sensing, bacteria can communicate with each other at high cell densities via autoinducers
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