MICR3330 Lecture 09: Repressors and Activators 4/29/24

The following are examples of what?

-Global responses
-Sigma factors
-One component systems
-Two component systems

Alteration of gene expression for survival

What type of transcriptional factor is this?

-Modifies how RNA polymerase + sigma factor bind to promoter
-Proteins bind to DNA motifs within -35/-10 box promoter region
-Activates or represses transcription

Trans

Name the two domains of activators and repressors

1) DBD (DNA-binding)
2) EBD (Effector-binding)

Name this domain of a modular activator/repressor that binds to a ligand, causing a conformational change

EBD

Name this domain:

-Conformationally changed when a ligand binds
-5 different families
-Includes Fur, PerR, and Zur (with metal ion ligands)
-One component systems

DBD

What type of system is this?

-Regulators dimerize
-Monomers bind to DNA alpha helix
-Protein-DNA interaction does not disrupt base pairing
-Bases bond noncovalently
-Homodimers have same consensus sequence (inverted repeat)

One component (DBD)

What type of system is this?

-Very diverse
-Protein domain binds co-factors
and responds to pH/temperature
-Evolved from transport protein molecule-binding domains
-Can either recognize one (specialized) or multiple compounds (generalized)
-Simple feedback loop: without co-factor, regulator is inactive and cannot bind DNA

One component (EBD)

What is the purpose of having multiple EBDs linked to the same DBD?

-Multiple signals at once
-Improves sensitivity
-Array of the same domains

Name this alpha helix which binds DNA

Recognition/sequence helix

Name the consensus sequence of a recognition helix

Operator motif

Name the four steps of transcription

Hint: B, I, I, E

1) Binding (RNAP)
2) Isomerization (Sigma Factor)
3) Initiation (RNAP)
4) Elongation (RNAP)

Name this protein:

-Holoenzyme
-5 subunits
-Initiates transcription

RNA Polymerase (RNAP)

Name this protein:

-Forms closed complex (Binding)
-Separates DNA strands and forms open complex (Isomerization)

Sigma factor

Name this protein:

-Dissociates after initiation of transcription

Sigma factor

Name this protein:

-Continues elongation after sigma factor has dissociated

RNA Polymerase (RNAP)

What type of one-component system RNAP repression is this?

-The repressor binds the promoter sequence
-Sigma factor is blocked from binding

Steric hindrance

What type of one-component system RNAP repression is this?

-Blocks +1 site
-Initiation of mRNA synthesis is inhibited

Roadblock

What type of one-component system RNAP repression is this?

-Inhibits binding of RNA polymerase to the promoter

Deformation

What type of one-component system RNAP repression is this?

-RNA polymerase is inhibited
-Cannot transition from closed to open complex
-Stops dsDNA from being melted

Anti-activation

What type of one-component system RNAP repression is this?

-Binding to alpha subunit

Inhibition of clearance from promoter

What component does one-component system de-repression rely on?

Ligand-binding status of EBD

What is the result of this one-component system de-repression?

-Repressor binds to DNA in absence of DNA

Ligand binds to EBD domain, DBD releases promoter

What is the result of this one-component system de-repression?

-Ligand binds EBD domain
-Repressor binds promoter

In absence of ligand, DBD domain releases promoter

How do repressor proteins undo negative regulation of transcription?

1) Ligand binds to repressor, releases it from DNA
2) Ligand is removed, repressor is released from DNA

What type of one-component RNAP mode of activation is this?

-Activator binds to a sequence upstream of promoter
-Interacts with alpha domain
-Stabilizes closed complex formation

-Class I
-Better binding, high transcription rates

What type of one-component RNAP mode of activation is this?

-Activator binds sequence close to promoter
-Enhances ability to melt dsDNA strands

-Class II
-Stabilizes RNAP open complex formation

What type of one-component RNAP mode of activation is this?

-Activator binds promoter region without optimal spacing
-Activator changes shape in presence of ligand

-Conformational change
-DNA is contorted
-Brings -10 and -35 boxes closer for optimal binding by RNAP

What type of one-component RNAP mode of activation is this?

-NtrC bends DNA to form stable closed complex
-Hydrolyzes ATP

-Activation of Sigma 54 promoters
-Initiates open conformational change in sigma factor

Name this protein:

-Bends DNA and hydrolyzes ATP
-Initiates open conformational change in sigma 54

NtrC

What type of sigma promoter is activated after NtrC creates an open conformation?

Sigma 54 Promoter

What type of system is this?

-Signal transduction system
-Free cytoplasmic component + transmembrane component

Two-component system

Would you expect a system that includes signal transduction to include one or two components?

Two-component system

Which part of the two-component system is this?

-Sensor kinase
-Three domains (periplasmic, membrane, cytoplasmic)

Transmembrane component

What is the function of this two-component transmembrane domain?

-Periplasmic domain

-Senses stimulus

Where is this two-component transmembrane domain found?

-Senses stimulus

Periplasmic domain

What is the function of this two-component transmembrane domain?

-Membrane domain

-Contains conserved histidine residue
-Dimerization domain

Where is this two-component transmembrane domain found?

-Contains conserved histidine residue
-Dimerization domain

Membrane domain

What is the function of this two-component transmembrane domain?

-Cytoplasmic domain

-Histidine kinase
-Depends on signal from periplasmic domain

Where is this two-component transmembrane domain found?

-Contains histidine kinase that phosphorylates dimerization of histidine residue
-Dependent on signal from periplasmic domain

Cytoplasmic domain

True or false: The conserved histidine residue can be phosphorylated by any type of kinase

False, it can only be phosphorylated by the histidine kinase in the cytoplasmic domain

Which part of the two-component system is this?

-Response regulator
-Has DBD and EBD domains
-DBD conformation is controlled by EBD receiver domain
-EBD receiver domain contains conserved aspartate residue
-Requires histidine kinase from sensor kinase to phosphorylate receiver domain

Cytoplasmic component (free)

Name this domain:

-Cytoplasmic domain of two-component system
-Includes aspartate residue
-When phosphorylated by histidine kinase, causes DBD conformational change

EBD receiver domain

What type of conserved residue does the two-component EBD receiver domain contain?

Aspartate residue

What type of conserved residue does the two-component sensor kinase contain?

Histidine residue

How do the two components of a two-component system interact?

1) Transmembrane component senses stimulus, phosphorylates histidine and undergoes dimerization
2) Cytoplasmic EBD receiver domain is phosphorylated by sensor kinase and DBD undergoes conformational change and gene expression

Which component of the two-component system is this?

-Senses stimulus
-Phosphorylates dimerization domain of conserved Histidine residue
-Donates phosphate group to EBD receiver domain

Histidine kinase (HK)

Which component of the two-component system is this?

-Modular organization (similar to one-component systems)
-EBD receiver domain contains conserved aspartate residue
-DBD Conformation is determined by aspartate phosphorylation state

Response Regulator (RR)

What is the result of this negative feedback loop?

-Environmental stimulus is removed
-Histidine kinases become phosphatases
-Phosphates are removed from aspartate residues in response regulator

Two-component system is shut off

What causes the histidine kinases to become phosphatases and remove phosphate groups from the Aspartate residues?

Environmental stimulus is removed

Name this protein:

-Becomes phosphatase
-Removed phosphates from aspartate residues in absence of environmental stimulus

Histidine kinase

What type of proteins are these?

-CAMPs
-Heme, O2
-cAMP
-Very diverse

Histidine kinases

Which part of the histidine kinases is relatively conserved?

Histidine kinase domain

What type of protein is this?

-REC
-Receiver and five DBD domains are all conserved

Response regulators

True or False: Some response regulators can be phosphorylated by multiple histidine kinases

True

What type of pathogen is this?

-Originates in water/soil environment
-After being ingested, uses iron to sense the host

Escherichia coli

What is the result of this two-component system process?

-Presence of high iron (Fe3+)
-PrmB- histidine kinase transfers phosphae from dimerization domain to response regulator PrmA
-PrmA receiver domain is phosphorylated and initiates transcription of ArnT and EptA transferases

Lipid A loses its negative charge and makes bacteria resistant to positive defensins

What initiates gene transcription of Lipid A transferases such as ArnT and EptA?

PmrA must be phosphorylated by PmrB-

What is the function of PmrB?

Histidine kinase

What is the function of PmrA?

Response regulator

Which component is responsible for initiating transcription of ArnT and EptA after being phosphorylated?

PmrA

Which component is responsible for phsophorylating the response regulator?

PmrB-

What is the function of ArnT and EptA?

-Transferases
-Removes lipid A negative charge
-Adds arabinose and phosphoethanolamine

Name the two genes that are responsible for adding arabinose and phosphoethanolamine to Lipid A

-ArnT
-EptA

Why do bacteria that possess PmrA/B- systems have access to high iron?

They invade bloodstream (high iron environment)

What innate defenses do bacteria need to protect themselves against using high iron?

Defensins

Name this protein:

-cAMPs: Cationic antimicrobial peptides
-Positively-charged
-Bind to negatively-charged Lipid A

Defensins

How do bacteria become resistant to defensins and grow in the blood?

-Negative Lipid A is modified with arabinose or phosphoethanolamine
-Lipid A is no longer negative
-Positive defensins are cAMPs, can no longer bind Lipid A to target bacteria

What is the purpose of these proteins?

-Arabinose
-Phosphoethanolamine

-When added to Lipid A, removes negative charged
-Allows bacteria to resist defensins and grow in blood

Which system has a larger number of systems at higher genome sizes? One or two component systems?

One-component system

True or False: The larger the genome, the greater number of regulatory systems necessary for life

True

True or False: There is a conserved set of regulatory proteins that control responses to environmental stimuli (oxygen, osmotic stress, heat)

True

True or False: All networks of genes controlled in each bacterium in response to a stress signal are the same

False: There are differences between the stress responses of E. coli and Neisseria gonorrhea

Which stress response is this?

-Proteins are synthesized in unfolded state
-Chaperones assist with folding to change native conformation
-Stress signals result in misfolded/aggregated proteins
-Regulated by sigma factors 32 (RpoH) and sigma factor E (RpoE)

E. coli

Which stress response bacteria is this?

-Versatile (environmental and pathogenic, lives in gut epithelium)
-4 Mb genome
-30 two-component systems
-230 one-component systems
-7 sigma factors

E. coli

Which stress response bacteria is this?

-Obligate human pathogen in human genital tract
-Spread by genital secretions that cause infertility in 1/3 of women
-Simple signal transduction system
-2 Mb genome
-3 two-component systems
-30 one-component systems
-3 sigma factors

Neisseria gonorrhea

Which bacterial stress response are these sigma factors associated with?

-RpoD
-RpoE
-RpoH

Neisseria gonorrhea

What is upregulated during heat shock in E. coli and N. gonorrhea?

RpoH

What part of bacterial response regulators allows them to be flexible/diverse despite conservation?

-Genes present in bacterial chromosome
-Genes with appropriate promoters

What causes diversity in bacterial response regulator pathways to similar stress signals?

-Integration of different signals