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MICR3330 > 9 - Protein DNA interactions (Repressors, activators) > Flashcards

9 - Protein DNA interactions (Repressors, activators) Flashcards

1
Q

Conditions that may challenge bacteria

A
  • Nutrient availability
  • Antibiotics
  • Toxins
  • Temperature
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2
Q

Alteration of gene expression to survive (global responses)

A
  • Sigma factors
  • One component systems
  • Two component systems
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3
Q

Mechanisms controlling transcription

A
  • Sigma factors
  • Transcriptional regulatory factors
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4
Q

Transcriptional factors

A
  • 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)
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5
Q

Transcriptional regulators of one component systems

A
  • 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
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6
Q

Diversity of DBDs

A
  • 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
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7
Q

Mechanism of DBDs

A
  • 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)
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8
Q

DBDs operator motif

A

Consensus sequence

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9
Q

Diversity of EBDs

A
  • 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)
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10
Q

Functions of EBDs

A
  • Specialised (recognise only one molecule)
  • Generalist (recognise multiple related compounds)
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11
Q

Versatility of one component systems

A

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

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12
Q

Flexibility of one component systems

A

can be activators, repressors, or both

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13
Q

Initiation of transcription

A
  • RNA polymerase 5 subunit holoenzyme
  • Closed complex formed
  • Isomerisation forms open complex
  • RNA synthesis begins (initiation)
  • SIgma factor dissociates
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14
Q

Closed complex

A

Forms when sigma factor binds promotor

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15
Q

Open complex

A

Forms when sigma factor separates the dsDNA strands

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16
Q

Different modes of repression in one component systems

A
  • Steric hinderance
  • Roadblock
  • Deformation
  • Anti-activation
  • Inhibition of clearance from promoter
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17
Q

Steric hinderance

A

The repressor binds the promoter sequence and blocks sigma factor binding

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18
Q

Roadblock

A

Inhibition of initiation of mRNA synthesis at the +1 position by blocking this site

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19
Q

Deformation

A

Inhibits binding of RNA pol to the
promoter

20
Q

Anti activation

A

Inhibition of RNA Pol transition from closed to open ie. stops melting dsDNA

21
Q

Inhibition of clearance from the promotor

A

By binding to the alpha subunit

22
Q

Relieving repression of one component systems

A
  • De repression
  • Relies on the ligand binding status of the EBD
  • Two scenarios
23
Q

Scenario 1 of relieving repression

A
  • The repressor is bound to the
    DNA in the absence of ligand
  • When ligand binds EBD domain
    the DBD releases the promoter
24
Q

Scenario 2 of relieving repression

A
  • The ligand binds the EBD domain and the repressor binds the promoter
  • In the absence of ligand, the DBD
    domain releases the promoter
25
Q

Mode of activation of one component systems

A
  • Class 1
  • Class 2
  • Conformational change
  • Activation of sigma 54 promoters
26
Q

Class 1 activation

A

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)

27
Q

Class 2 activation

A

Activator binds a sequence close to the promoter, enhances the ability to melt apart dsDNA strands (stabilise open complex formation of the RNApol)

28
Q

Conformational change activation

A
  • 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
29
Q

Activation of sigma 54 promoters

A
  • 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
30
Q

Two component systems

A

Signal transduction system (one component spans the plasma membrane, the other is free in the cytoplasm)

31
Q

Parts of the component that spans the plasma membrane in a two component system

A
  • Sensor kinase
  • Three domains
32
Q

Three domains of component that spans plasma membrane

A
  • 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)
33
Q

Component that is free in cytoplasm in two component system

A
  • 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
34
Q

Histidine kinase (HK)

A
  • Senses stimulus resulting in phosphorylation of the dimerization domain at the conserved His residue
  • Donates phosphate group to receiver domain of response regulator
35
Q

Response regulator

A
  • 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
36
Q

Feedback loop of two component systems

A

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

37
Q

Diversity of histidine kinases (two component system)

A
  • Sensing domains are very diverse
  • Kinase domain relatively conserved
38
Q

Diversity of response regulators (two component systems)

A

Receiver and DBD domains (5 domains) are conserved

39
Q

Diversity of two component system

A
  • Single or multiple inputs reflecting lifestyle
  • Some response regulators can be phosphorylated by multiple histidine kinases
40
Q

Example of two component systems

A

PmrA/B - using iron to send the host

41
Q

PmrA/B

A
  • 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
42
Q

ArnT and EptA

A

Transferases that change the structure of the lipid A by adding
arabinose and phosphoethanolamine

43
Q

Why do bacteria change the structure of lipid A

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
44
Q

Distribution of regulatory proteins in bacteria

A
  • Depends on size of chromosome and lifestyle
  • The more complicated lifestyle, the larger the genome, the greater number of regulatory systems necessary for life
45
Q
A

MICR3330 (29 decks)

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