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MICR3330 > 8 - Intro to Bacterial Gene Regulation > Flashcards

8 - Intro to Bacterial Gene Regulation Flashcards

1
Q

Central dogma

A

DNA –Transcription–> RNA –Translation–> Protein

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2
Q

Gene naming convention

A

lower case, italics (e,g, rpoH)

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3
Q

Protein naming convention

A

Uppercase, no italics (RpoH)

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4
Q

Outcomes of transcription

A

tRNA, mRNA, rRNA

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5
Q

Regulon

A

Multiple genes in different locations controlled by the same type of promoter thus resulting co-ordinated expression

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6
Q

Operon

A

Multiple genes in the same location, controlled by a single promoter

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7
Q

Gene

A

Entire nucleic acid sequence necessary for expression of a gene product

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8
Q

Why do bacteria regulate gene expression

A

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

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9
Q

Examples of global responses

A
  • SOS response (sudden global DNA damage)
  • Starvation response
  • Heat stress response
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10
Q

Specific responses

A
  • lac operon to utilise lactose as an energy source
  • trp operon to synthesise tryptophan
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11
Q

Hierarchical control mechanisms for gene expression

A
  • Mechanisms controlling transcription (e.g. sigma factors)
  • Mechanisms for controlling translation (e.g. occlusion of the Shine Dalgarno sequence)
  • Mechanisms for controlling protein function (e.g. sequestration)
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12
Q

Mechanisms controlling gene expression at the transcriptional level

A
  • Different promoters bind different sigma factors of RNA polymerase
  • DNA binding proteins bind the promoter region acting as repressors or activators of transcription
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13
Q

5 subunits of RNA polymerase holoenzyme

A
  • 2 large subunits β and β’
  • 2 copies of smaller α
  • 1 copy of σ^70
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14
Q

σ^70

A
  • Initiation factor
  • Interacts with promoter
  • Binds to specific sequences near -10 and -35 boxes
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15
Q

α

A

Controls frequency of initiation of transcription

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16
Q

β and β’

A

Polymerase NTPs, transcribe DNA

17
Q

Sigma Factors

A
  • Form a reversible, reusable complex with RNA polymerase and aids promoter selectivity
  • Released before translation
  • Each sigma factor has a different affinity
    for RNA polymerase
  • Increased sigma factor concentration
    results in increased amplitude of
    expression from those genes under this control
18
Q

Different classes of sigma factors

A
  • σ^70 family (RpoD)
  • σ^54 family (RpoN)
19
Q

Different groups of σ^70 family

A
  • Group 1: Essential (RpoD)
  • Group 2: Non-essential primary like sigma factors (stationary phase σ factors e.g. σ38 or RpoS)
  • Group 3: Alternative factors (heat shock σ factors e.g. σ32 or RpoH)
  • Group 4: RpoE subfamily (ECF σ factors)
20
Q

Domains of σ^70

A
  • S1: Only in σ70, auto-inhibition of FNA binding determinants
  • S2: Interacts with -10 of promoter
  • S3: Three helix domain
  • S4: Interact with -35 of promoter
21
Q

When are minor sigma factors expressed

A

In response to physiological signals such as starvation, temperature, growth phase

22
Q

What is the strength of sigma factor binding determined by

A

The spacing of the -10 and -35 regions

23
Q

RpoH

A
  • Heat stress proteins have RpoH promoter sequences
  • Under normal conditions, RpoH expression is low therefore, the expression of RpoH controlled regulon is low
24
Q

What happens to RpoH under heat stress

A
  • RpoH expression increases, therefore the expression of RpoH controlled regulon increases
  • The cell has the right concentration of proteases and chaperones to remove mis-folded proteins so it can survive heat shock
  • When temperature decreases the expression of RpoH declines to baseline levels
25
Q

How is RpoH regulated in E. coli

A
  • Post-translational level (sequestration and
    degradation)
  • Post-transcriptional (mRNA structure prevents translation)
  • Transcriptional level (promoter recognised by different sigma factors for induced expression)
26
Q

RpoH expression during normal growth

A
  • Low levels of RpoH protein is translated
  • Low levels of DnaJ/DnaK/GrpE aretranslated. These form a complex in the inner membrane
  • FtsH (a protease) associates with this complex and degrades proteins bound to DnaJ/DnaK/GrpE
  • Most RpoH is bound to DnaK/DnaJ/GrpE and FtsH degrades RpoH
  • Therefore, levels of RpoH are kept low
27
Q

Thermosensitive riboswitch

A
  • At normal temperature, the mRNA forms a tertiary structure (thermosensitive riboswitch)
  • Improves mRNA stability and occludes the Shine Dalgarno (SD) site to prevent translation
  • Triggers degradation (mRNA half life of 40secs) so RpoH is low
28
Q

RpoH during heat stress

A
  • During heat shock, the structure of the mRNA unwinds
  • allows ribosome access to the SD site
  • Half life of mRNA increases to 4 mins
  • Increased translation of RpoH
  • Proportion of RNA pol association with RpoH increases, therefore induction of the genes encoding factors that protect from stress
29
Q

DnaK/DnaJ/GrpE chaperone complex

A
  • Preferentially recognizes misfolded proteins in the cytoplasm
  • Associates with ClpB foldase that re-folds proteins to correct conformation
30
Q

RpoH degradation cycle feedback loop

A
  • Once misfolded proteins are removed, RpoH binds to DnaK/DnaJ/GrpE
  • RpoH levels decline in the cytoplasm and proportionally the amount of sigma factor
    binding RNA polymerase declines
  • So decreased transcription from all RpoH-dependent promoters
31
Q

Where does RpoH maintain protein folding system

A

Cytoplasm

32
Q

What is responsible for the protein repair response in the periplasm

A

RpoE

33
Q

RpoE in E. coli

A
  • Responds to misfolded proteins in the periplasm
  • Gram negative bacteria
  • Must transmit response signal from periplasm to cytoplasm compartments across the periplasmic membrane
34
Q

RpoE under normal growth conditions

A
  • Sigma E is sequestered to the inner membrane via RseA and RseB
  • This complex sits in the cytoplasmic membrane
  • The cytoplasmic domain binds to RpoE so only low levels of RpoE associate with RNA polymerase
  • DegS is a protease that remains inactive at normal temperature as it folds upon itself by the PDZ domain
35
Q

RpoE during stress

A
  • DegS unfolds, becomes active and degrades the mis folded proteins
  • RpoE is released from RseA and RseB by YaeL protease
  • RpoE associates with RNA polymerase and induces the stress response from RpoE dependent promoters including RpoH
  • If the response is to heat, the increased mRNA from RpoH will unwind and will be translated (both systems work together)
  • Once all misfolded proteins are removed, RpoE re-associates with RseAB, shutting the response off
36
Q

Effectors that alterations to folding dependent on

A
  • Metabolites
  • tRNAs
  • Temperature
37
Q

What does folding of the 5’ UTR of mRNA influence

A
  • Continuation of transcription
  • mRNA half life
  • Translation

MICR3330 (29 decks)

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