Transcript Bio3124 Lecture 10

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
– Cytoplasmic response regulator
Takes phosphate from sensor
 Binds chromosome
 Alters transcription rate of
multiple genes

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 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

b-galactosidase
converts lactose to
glucose and
galactose
People also make bgalactosidase
 If not, person is lactoseintolerant

The E. coli lac Operon
• The lacZ gene encodes b-galactosidase
• The lacY gene encodes lactose permease
– Need both proteins to digest lactose
• Operon
– Multiple genes transcribed from one promoter
The E. coli lac Operon
• Repressor protein LacI
blocks transcription
– Repressor binds to
operator
– Blocks s factor from
binding promoter
• 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 Operon by 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
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
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: 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.
Transcriptional Attenuation Mechanism of the trp Operon
High tryptophan
Low tryptophan
Animation: Transcriptional Attenuation
Sigma Factor Regulation
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s factors regulate transcription of all genes
Recognize promoter sequences differentially
Specific to a subset of genes
Direct RNA Pol to start transcription
Alternative s factors used for global transcriptional
regulation of related genes
How are sigma factors regulated?
• Temperature-sensitive mRNA 2°-structure
– at 42°C s70 degraded rapidly
– Allows translation of s32 (sH) only at high temperature
• Synthesis of proteins that
inhibit s factors
– Anti-s factors block s activity
until needed
– Anti-anti-s 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
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
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 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
Quorum Sensing: Bioluminescence induction
Animation: Quorum Sensing
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 operon is regulated as follows:
- Operon is off when LacI binds to the operator.
- Operon is on when allolactose binds to LacI; cAMPCRP 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
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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
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
●