9 - Protein DNA interactions (Repressors, activators) Flashcards
Conditions that may challenge bacteria
- Nutrient availability
- Antibiotics
- Toxins
- Temperature
Alteration of gene expression to survive (global responses)
- Sigma factors
- One component systems
- Two component systems
Mechanisms controlling transcription
- Sigma factors
- Transcriptional regulatory factors
Transcriptional factors
- Modify how the RNA polymerase + sigma factor binds to the promoter region
- Trans-acting proteins that bind to motifs of the DNA within the promoter region
- Their binding to DNA motifs is controlled by their 3D structure which is influenced by co-factors
- Act as activators (increasing transcription) or repressors (preventing transcription)
Transcriptional regulators of one component systems
- DNA Binding Domain (DBD) and effector binding domain (EBD)
- Act ‘in trans’ to either activate or repress transcription initiation from the promoter by interaction with RNA polymerase
- Divided into 16 superfamilies
Diversity of DBDs
- Five different DBD families in bacteria
- Different members of the same DBD family bind different operator sequences
- Specificity is determined by different amino acids in the helix and their interaction with the DNA nucleotides
Mechanism of DBDs
- One component system regulators dimerise
- Each monomer of the dimer binds
adjacent major grooves of the DNA double helix - The α-helix which binds the DNA is called the recognition helix (or sequence reading helix)
- Protein-DNA interaction does not disrupt the base pairing of the DNA (Bonds with bases non-covalently)
DBDs operator motif
Consensus sequence
Diversity of EBDs
- Very diverse
- Protein domain that binds co factors (small molecules) or respond to environmental triggers (eg temperature, pH).
- Forms a simple feedback loop-
when co-factor is not around,
the regulator is inactive and
cannot bind DNA (no transcription = no pump of molecule out of cell)
Functions of EBDs
- Specialised (recognise only one molecule)
- Generalist (recognise multiple related compounds)
Versatility of one component systems
Can have one or multiple EBDs linked to one DBD so the the response has multiple signals at once and improved sensitivity by having an array of same domains
Flexibility of one component systems
can be activators, repressors, or both
Initiation of transcription
- RNA polymerase 5 subunit holoenzyme
- Closed complex formed
- Isomerisation forms open complex
- RNA synthesis begins (initiation)
- SIgma factor dissociates
Closed complex
Forms when sigma factor binds promotor
Open complex
Forms when sigma factor separates the dsDNA strands
Different modes of repression in one component systems
- Steric hinderance
- Roadblock
- Deformation
- Anti-activation
- Inhibition of clearance from promoter
Steric hinderance
The repressor binds the promoter sequence and blocks sigma factor binding
Roadblock
Inhibition of initiation of mRNA synthesis at the +1 position by blocking this site
Deformation
Inhibits binding of RNA pol to the
promoter
Anti activation
Inhibition of RNA Pol transition from closed to open ie. stops melting dsDNA
Inhibition of clearance from the promotor
By binding to the alpha subunit
Relieving repression of one component systems
- De repression
- Relies on the ligand binding status of the EBD
- Two scenarios
Scenario 1 of relieving repression
- The repressor is bound to the
DNA in the absence of ligand - When ligand binds EBD domain
the DBD releases the promoter
Scenario 2 of relieving repression
- The ligand binds the EBD domain and the repressor binds the promoter
- In the absence of ligand, the DBD
domain releases the promoter
Mode of activation of one component systems
- Class 1
- Class 2
- Conformational change
- Activation of sigma 54 promoters
Class 1 activation
Activator binds to a sequence distantly upstream of the promoter, interacts with the alpha domain and stabilises closed complex formation (better binding, high transcription rates)
Class 2 activation
Activator binds a sequence close to the promoter, enhances the ability to melt apart dsDNA strands (stabilise open complex formation of the RNApol)
Conformational change activation
- Activator binds promoter region without optimal spacing, activator changes shape in the presence of a ligand
- Results in contortion of the DNA
bringing the -10 and -35 boxes to optimal distance for better binding by RNA pol
Activation of sigma 54 promoters
- Bends DNA to form a stable closed complex
- eg. NtrC bends DNA, hydrolyses ATP to initiate conformational change in sigma factor to move to the open conformation
Two component systems
Signal transduction system (one component spans the plasma membrane, the other is free in the cytoplasm)
Parts of the component that spans the plasma membrane in a two component system
- Sensor kinase
- Three domains
Three domains of component that spans plasma membrane
- Periplasmic domain sensing the stimulus
- Membrane domain (a dimerisation domain containing a conserved histidine residue)
- Cytoplasmic domain (always a histidine kinase that phosphorylates the dimerisation histidine residue dependent upon the signal from the periplasmic
domain)
Component that is free in cytoplasm in two component system
- Response regulator
- This protein has a DBD and a specialised EBD called a receiver domain
- Receiver domain has a conserved
aspartate residue which is phosphorylated by the histidine kinase of the sensor kinase - This event controls the conformation of the DBD
Histidine kinase (HK)
- Senses stimulus resulting in phosphorylation of the dimerization domain at the conserved His residue
- Donates phosphate group to receiver domain of response regulator
Response regulator
- Modular organisation similar to one component systems
- Conformation of the DNA binding
domain (DBD) is determined by phosphorylation state of receiver
domain at the conserved aspartate
residue
Feedback loop of two component systems
When environmental stimulus is gone, HKs become phosphatases that remove the phosphate from the conserved Asp residue in the RR, thus shutting the system off
Diversity of histidine kinases (two component system)
- Sensing domains are very diverse
- Kinase domain relatively conserved
Diversity of response regulators (two component systems)
Receiver and DBD domains (5 domains) are conserved
Diversity of two component system
- Single or multiple inputs reflecting lifestyle
- Some response regulators can be phosphorylated by multiple histidine kinases
Example of two component systems
PmrA/B - using iron to send the host
PmrA/B
- PmrB: histidine kinase that responds to high iron by transferring phosphate from the dimerisation domain to the receiver domain of PmrA
- Phosphorylated PmrA response regulator initiates transcription of many genes including ArnT and EptA
ArnT and EptA
Transferases that change the structure of the lipid A by adding
arabinose and phosphoethanolamine
Why do bacteria change the structure of lipid A
- Defensins are cationic antimicrobial peptides (CAMPs)
- CAMPs are positively charged and will bind to the negatively charged lipid A
- Modifying lipid a with arabinose or phosphoethanolamine removes this negative charge
- Bacteria become resistant to
defensins and can start growing in the blood
Distribution of regulatory proteins in bacteria
- Depends on size of chromosome and lifestyle
- The more complicated lifestyle, the larger the genome, the greater number of regulatory systems necessary for life
