MICR5831 L4: Introduction to Bacterial Gene Regulation 8/12/25

Explain how a repressor and activator protein works to control gene expression. (slide 6)

-Activators/repressors bind to ligands called co-repressors or inducers

-Ligands: small molecules like amino acids, sugars

-Respond to environmental changes such as pH, temperature

Describe 4 ways a repressor can prevent transcription of a gene (slide 7)

1) Repressor protein binds to the promoter, blocks the Sigma Factor

2) Repressor binds upstream of Promoter

3) Repressor binds opposite of RNAPol

4) RNApol binds Promoter but Repressor proteins stops the initiation phase

Describe 2 ways that an activator can induce transcription (slide 8)

1) Activator proteins stabilize the interaction of RNApol with the promoter

-Bending the DNA at the -35 and -10 sites

-Optimal distance for Sigma Factor

2)

-Activators bind a ligand that changes their conformation

-Enables them to bind the DNA operator

Explain how a regulatory protein can bind DNA? (slide 9)

1) Ligand binds to the EBD

2) DBD conformation changes

3) DBD will either release the DNA or bind to it more tightly

How does a regulatory protein sense environmental cues inside and outside the bacterial cell (slide 9 and 10)

1) Ligand enters cytoplasm to interact with the regulatory protein EBD

2) Histidine Kinase (HK) periplasmic domain binds ligands in periplasm, phosphorylates RR receiver domain

3) Response Regulator (RR) DBD domain is phosphorylated and acts as repressor/activator

Explain a simple two component circuit for gene expression (slide 10)

1) HK periplasmic domain binds ligands

2) His residue in HK dimerization domain is phosphorylated

3) HK phosphorylates Asp residue of the RR receiver domain, affecting DBD

4) DBD is phosphorylated and changes conformation into repressor/activator

Using an example, explain how a regulatory protein can be both an activator and repressor? (slide 11)

Without ligand arabinose:

-AraC forms an elongated dimer form

-Binds to ara02 and aral1, creates a loop,

-Prevents RNA pol from binding to the promoter

With the ligand arabinose:

-Compact dimer forms that binds aral1 and aral2

-RNApol binds to the araB promoter

-Transcription = catabolism of arabinose for carbon

-Another regulatory protein, CRP, also binds and this inhibits araC expression

Explain how attenuation works to control gene expression (slide 12)

1) Binding of Trp to the aporepressor makes holorepress holorepressor

2) Binding of holorepressor to the trp operator inhibits but does not altogether prevent-transcription

3) It lowers expression 100x fold but does not turn it off completely as the repressor binding is weak

What are the important features in the attenuator region? (slide 13)

Two hairpin loops:

Stem loop 1:2

-Contains a short ORF

-"Leader peptide coding region"

-Rich in Trp codons

Attenuator hairpin loop (stem 3:4)

-Ends in a U-rich transcriptional termination motif

-High Trp: Transcription elongation into trp operon cannot occur

-Low Trp: Entire operon is transcribed

How does coupling of transcription and translation result in attenuation (slide 14)

-Transcription is initiated

-First140 bp of the 5'mRNA are synthesised

-Ribosome binds the Shine Dalgarno site

-Transcription and translation are coupled

-Attenuator loop stops Trp transcription during High Trp

-Anti-terminator loop helps elongate mRNA during Low Trp

Using an example, explain how mRNA stability can be used to control gene expression (either slide 16 or slide 19)

1) mRNA stability affects RpoH expression

Normal temperature:

-mRNA forms thermosensitive riboswitch

-Occludes SD site to prevent translation

-Triggers degradation, mRNA half-life of 40secs

-RpoH is low

2) sRNA stability affects succinate dehydrogenase expression

Low Iron:

-Fur repressor de-repressors Enterochelin (iron scavenger) and rhyB promoters

-SRNA rhyB binds to succinate dehydrogenase mRNA

-Enables the RNAase to bind the mRNA

-Rapidly degrade the mRNA, shutting down the expression of the enzyme

Explain how RpoH levels are controlled during normal growth conditions. (slide 16 and 17)

Normal Temp:

1) mRNA forms thermosensitive riboswitch

2) Occludes SD site to prevent translation

3) Triggers degradation, mRNA half-life of 40secs

4) RpoH is low

Normal Growth:

1) rpoH is transcribed from a sigma 70 promoter

2) RpoH binds DnaK/DnaJ/GrpE in the inner membrane

3) Associates with FtsH protease which degrades RpoH

4) Low RpoH protein levels

Explain how RpoH levels are controlled during heat shock (slide 16 and slide 18)

mRNA stability is determined by secondary structure and sensitivity to degradation:

1) mRNA structure unwinds

2) Allows ribosome access to the Shine-Dalgarno site

3) Half-life of mRNA increases to 4 min

4) Increased translation of RpоH

5) Proportion of RNA pol association with RpoH increases

6) Induces genes encoding factors that protect from stress

De-repression during Heat Shock:

1) DnaK/DnaJ/GrpE chaperone preferentially recognizes misfolded proteins in the cytoplasm

2) Associates with ClpB foldase that refolds proteins to correct conformation

3) RpoH is released from DnaK/DnaJ/GrpE chaperone

4) RpoH associates with RNA polymerase

5) Increased transcription from RpoH-dependent promoters

What is a sRNA and what is one of its roles in controlling gene expression (slide 19)

-Anti-sense RNA encoded in intergenic spaces of the genome

-Controlling plasmid and viral replication, transposition, response to environmental cues

How does slipped strand mispairing occur in a bacterial cell? How does this affect gene expression (slide 20)

-Occurs during DNA replication when one or the other strand, slips and mis-pairs with the wrong repeat on the other strand

-Can alter the number of repeat units in a gene and lead to variations in protein expression

Using an example, explain the principles behind our understanding of orthologous regulatory circuits in different bacterial species (slide 21-22).

-Orthologues of regulatory factors retain core target genes but have captured other species specific genes

-PhoPQ and PmrAB live in different environments where low Mg and low Fe occur separately or together and species have adapted their pathways of regulation to suit these situations

Why do bacteria regulate gene expression?

To express a subset of proteins to permit the bacterium to survive current conditions

What are some bacterial examples of Global responses?

-SOS response

-Starvation response

-Heat stress response

What bacterial gene expression does global DNA damage initiate?

SOS response

What are some bacterial examples of Specific responses?

-lac operon: Utilize lactose as an energy source

-trp operon: Synthesize tryptophan

What are hierarchical control mechanisms for controlling transcription?

1) Sigma factor binding to RNA pol.

2) Transcriptional regulatory factors

3) Attenuation

What are hierarchical control mechanisms for controlling translation?

Occlusion of the Shine Dalgarno sequencе

What are hierarchical control mechanisms for controlling protein function?

1) Sequestration

2) Degradation

What ligands can activators and repressors bind to?

-Co repressors and Inducers

-Respond to pH and temperature changes

True or False: Co-repressors and Inducers are typically large molecules such as amino acids and sugars

False, they are small

True or False: Co-repressors and Inducers are typically small molecules such as amino acids and sugars

True

How does a repressor protein prevent Sigma factor from binding and initiating transcription?

Repressor protein binds to the promoter

How does a repressor protein prevent the elongation phase of transcription?

1) Repressor binds upstream of promoter

2) Repressor binds opposite of RNA polymerase

How does repressor protein stop the initiation phase?

Repressor protein blocks RNAPol after it binds the promoter

How do Activator proteins increase the frequency of initiation of transcription?

-Strong binding

-Stabilizes RNApol and promoter interaction

How do Activator proteins create a stronger bond between RNApol and the promoter?

-Conformational alteration of DNA

-Bend DNA so -10 and -35 are optimal distance apart

-Increases optimal binding by Sigma Factor

Why do Activators bend the DNA -10 and -35 sites in order to increase transcription initiation?

Bending the DNA creates an optimal distance for Sigma Factors to bind

True or False: Activators always bind a ligand that will change their conformation

True

What do Activators always bind a ligand that changes their conformation?

The conformational change enables them to bind the DNA operator and bend the DNA

What are the 2 parts of a Regulatory protein's modular structure?

1) DNA Binding Domain (DBD)

2) Effector Binding Domain (EBD)

Which part of a Regulatory Protein is this?

-Very conserved

-Only 5 types

DNA Binding Domain (DBD)

Which part of a Regulatory Protein is this?

-Binds a small molecule ligand

-Very diverse

Effector Binding Domain (EBD)

What type of Regulatory Protein domain can bind to the following small molecule ligands?

-Sugars

-Amino acids

-Antibiotics

-Metals

-Quorum molecules

Effector Binding Domain (EBD)

Which Regulatory Protein domain undergoes a conformational change as a result of a ligand binding to its counterpart?

DNA Binding Domain (DBD)

What happens to the DBD when a ligand binds to the EBD?

-Conformation changes

-Releases or binds more tightly to DNA

True or False: Ligands must enter the cytoplasm to interact with the Regulatory Protein and bind to the EBD

True

What type of cues do Activators and Repressors (EBD and DBD) use?

What type of cues do Two-Component Systems use?

1) Regulatory Proteins (EBD and DBD) use internal cues

2) Two-Component Systems use external cues

What are the two parts of a Two-Component System?

1) Histidine Kinase

2) Response Regulator

Where are these Two Components found? Hint: External cues.

1) Histidine Kinase (HK)

2) Response Regulator

1) Inner Membrane

2) Cytoplasm

Which part of a Two-Component System is this?

-Periplasmic domain binds ligands in the periplasm

-Triggers phosphorylation of the conserved His residue in the Dimerization domain

-Donates phosphate group to a conserved Aspartate residue of the Receiver domain of a response regulator

Histidine Kinase (HK)

What triggers phosphorylation of the conserved His residue in a Histidine Kinase's Dimerization domain?

Periplasmic domain binds a ligand in the periplasm

What happens when the conserved His residue of the Dimerization domain of a Histidine Kinase is phosphorylated?

Phosphorylates conserved Receiver Domain of Response Regulator

Which residues are phosphorylated in a Histidine Kinase and Response Regulator?

HK: His residue, Dimerization Domain

RR: Asp residue, Receiver Domain

Which part of a Two-Component System is this?

-Modular organization: DBD attached to a Receiver domain

-Conformation of the DBD is determined by phosphorylation state of Receiver domain

-Acts as a Repressor or Activator of gene expression

Response Regulator

What affects the conformation of the DBD in a cytoplasmic Response Regulator?

Receiver Domain phosphorylation status

What is this?

-Regulatory protein that regulates araBAD degradation operon

-Normally elongated dimer form binds araO2 and aral1

-With arabinose, forms compact dimer that binds ara1 and ara2

AraC

What is this?

-Arabinose degradation operon

-Transcription = arabinose catabolism

-Regulated by AraC and CRP regulatory proteins

-Includes araB promoter

araBAD

What is this?

-Promoter found on araBAD degradation operon

-Blocked by AraC in the absence of arabinose

-Increases arabinose catabolism when RNApol binds to it

araB

What happens to AraC in the absence of its ligand, Arabinose?

-AraC forms an elongated dimer form

-Binds to ara02 and aral1

-Creates a loop that prevents RNA pol from binding to the araB promoter of araBAD operon

What happens to AraC in the presence of its ligand, Arabinose?

-Compact dimer forms binds aral1 and aral2

-Allows RNApol to bind to the araB promoter

-CRP also binds and this inhibits araC expression

True or False: If arabinose is present, RNAPol will bind to araB promoter and increase catabolism of arabinose for carbon

True

What is this?

-Repressor protein that regulates tryptophan biosynthesis

TrpR

What is this?

-Co-repressor in the trp operon

-Binds to TrpR repressor, helps it bind DNA operator sequence

-Inhibits transcription of the trp operon

Tryptophan (Trp)

What is this?

-Inactive form of TrpR

-When Corepressor (Trp) is absent, cannot bind to operator site

-RNAPol can bind to promoter and initiate transcription

Aporepressor

What is this?

-Aporepressor + Corepressor complex

-Binds operator of Trp operon to prevent transcription

-Lowers expression 100x but has weak bond

Holorepressor

What happens when the Holorepressor binds to the operator of the Trp operon?

-Lowers expression 100x fold

-Does not turn it off completely

-Repressor binding is weak

How much does attenuation decrease the rate of trp transcription?

10x decrease

How much is the modulatory range of a holorepressor and attenuation on Trp operon?

100x10 = 1000x

What parts would you find upstream of the Attenuator site/region?

1) Promoter

2) Operator that TrpR binds

3) Leader sequence, first 140 bp of mRNA

What is this?

-First 140 bp of the mRNA

-Transcribed upstream of the start codon of trpE

Leader sequence

What part of the DNA sequence does TrpR bind to?

Operator

What is this?

-Two hairpin loops: Stem and Attenuator Hairpin

-High Trp: Transcription elongation into trp operon can't occur

-Low Trp: Entire trp operon is transcribed

Attenuator region

What hairpin loop found in the Attenuator region is this?

-Contains a short ORF called "leader peptide coding region"

-Rich in Trp codon

Stem loop (1:2)

What hairpin loop found in the Attenuator region is this?

-Ends in a U-rich transcriptional termination motif

Attenuator hairpin loop (stem 3:4)

What happens once transcription is initiated and the Leader sequence (first 140 bp of the 5' mRNA) have been synthesized?

-Ribosome binds Shine-Dalgarno site

-This means transcription/translation are coupled

True or False: Transcription and translation are coupled

True

What happens if there is High Trp?

1) Ribosome completes LP synthesis

2) Pause at the stop codon

3) Attenuator loop (3:4) forms, binds to RNApol

5) RNAPol pauses/releases DNA

6) Terminates transcription

What happens if there is Low Trp?

1) Ribosome pauses inside LP ORF due to lack of tRNA-Trp for translation

2) Anti-terminator looр (2:3) forms

3) mRNA elongation continues into trp operon.

What is this?

-mRNA secondary structure

-Signal for degradation and Thermostat

-Low protein/transcription at normal growth conditions

-High transcription during heat shock

rPOH

What happens to mRNA during normal temperature?

-mRNA forms a tertiary structure

-Thermosensitive riboswitch blocks Shine-Dalgarno site

-Prevent translation, triggers degradation

-mRNA half-life of 40secs, low RpoH

What happens to RpoH during normal growth conditions?

1) rpoH is transcribed from a Sigma 70 promoter

2) RpoH protein is bound to DnaK/DnaJ/GrpE in the inner membrane

3) Complex associates with FtsH protease

4) FtsH degrades RpoH

Normal growth conditions. Name the following:

1) Promoter that rpoH is transcribed from

2) Inner Membrane complex that RpoH protein is bound to

3) Protease that associates with RpoH and degrades it

1) Sigma 70

2) DnaK/DnaJ/GrpE

3) FtsH

True or False: High levels of RpoH transcription is required to make the DnaK/DnaJ/GrpE complex for protein refolding

False, very low levels of transcription from operons with RpoH-dependent promoters are needed

What happens to mRNA during heat shock?

-mRNA structure unwinds

-Allows ribosome access to the Shine-Dalgarno site

-Half-life of mRNA increases to 4 min

-Increased translation of RpоH

-Proportion of RNA pol association with RpoH increases

-Induces genes encoding factors that protect from stress

What structure does the ribosome need access to in order to increase translation of RPOH and half-life of mRNA?

Shine Dalgarno Site

What is the mRNA tertiary structure that blocks the SD site at normal temperature, resulting in low RpoH and a short mRNA half life?

-Thermosensitive Riboswitch

-Triggers degradation

True or False: RpoH is high during normal temperatures and low during heat shock

False

True or False: RpoH is low during normal temperatures and high during heat shock

True

What happens to RpoH repression during heat shock?

1) DnaK/DnaJ/GrpE chaperone preferentially recognizes misfolded proteins in the cytoplasm

2) Associates with ClpB foldase that refolds proteins to correct conformation

3) RpoH is released from DnaK/DnaJ/GrpE chaperone

4) RpoH associates with RNA polymerase

5) Increased transcription from RpoH-dependent promoters

Heat shock conditions apply. Name the following:

1) Chaperone that preferentially recognizes misfolded proteins

2) Enzyme that associates with chaperone and refolds proteins

3) What type of transcription is increased when RpoH associates with RNAPolymerase?

1) DnaK/DnaJ/GrpE

2) ClpB foldase

3) DnaK/DnaJ/GrpE, GroES/EL, ClpP and other heat stress genes from RpoH-dependent promoters

True or False: Chaperones are expressed less during heat shock conditions and deal with misfolded proteins to restore normal function

False

True or False: Chaperones are expressed more during heat shock conditions and deal with misfolded proteins to restore normal function

True

What is this?

-Anti-sense RNA

-Highly versatile regulatory functions

-Encoded in intergenic spaces of the genome

sRNA (Small)

What are some functions of small sRNA?

1) Plasmid/Viral Replication

2) Transposition

3) Response to Environmental Cues (rhyB)

What is this?

-Iron regulator

-Binds co-repressor Fe if high iron levels present

-Represses Enterochelin and rhyB

Fur

What is this?

-Scavenger protein, needed to scavenge iron

-Repressed by Fur iron regulator during high iron levels

Enterochelin

What is this?

-sRNA or anti-sense RNA

-Binds to mRNA of succinate dehydrogenase during low iron

-Repressed by Fur iron regulator and Fe co-repressor if high iron

rhyB

What is this?

-Uses iron for function

-Decreased expression when iron is low

-rhyB sRNA will bind to it so RNAase can degrade it

Succinate dehydrogenase

What environmental response does sRNA initiate during high levels of iron?

-Fur iron regulator binds its co-repressor Fe,

-Enterochelin is repressed, rhyB is suppressed

-Succinate dehydrogenase (which uses iron for function) is expressed

What environmental response does sRNA initiate during low levels of iron?

-Fur repressor releases the promoters of enterochelin and rhyB

-SRNA rhyB binds succinate dehydrogenase mRNA, creates complex

-RNAase binds and rapidly degrades mRNA

-Succinate dehydrogenase expression shut down

What is this?

-Phase variation resulting in stochastic expression of a gene in a population of daughter cells

-The progenitor cell may be "on," but progeny could be either "off" or "on" randomly

Slipped strand mispairing

When does mispairing occur in DNA replication?

One or the other strand slips and mis-pairs with the wrong repeat on the other strand

True or False: Mispairing repeats can be homopolymeric (single nucleotide repeats) or heteropolymeric (repeats of dimers, trimers, tetramers, etc)

True

What determines the frequency of slippage/slipped strand mispairing?

Length of the tract

-Longer tract, higher frequency of slippage

-Can be as high as 1 in 100 daughter cells

Name an example of a highly heterogeneous population that can be controlled/maintained by cell replication

-N. gonorrhea

-Has over 100 genes

-Gene expression controlled by slippage