8 - Intro to Bacterial Gene Regulation

Question 1

Central dogma

Answer 1

DNA –Transcription–> RNA –Translation–> Protein

Question 2

Gene naming convention

Answer 2

lower case, italics (e,g, rpoH)

Question 3

Protein naming convention

Answer 3

Uppercase, no italics (RpoH)

Question 4

Outcomes of transcription

Answer 4

tRNA, mRNA, rRNA

Question 5

Regulon

Answer 5

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

Question 6

Operon

Answer 6

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

Question 7

Gene

Answer 7

Entire nucleic acid sequence necessary for expression of a gene product

Question 8

Why do bacteria regulate gene expression

Answer 8

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

Question 9

Examples of global responses

Answer 9

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

Question 10

Specific responses

Answer 10

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

Question 11

Hierarchical control mechanisms for gene expression

Answer 11

• 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)

Question 12

Mechanisms controlling gene expression at the transcriptional level

Answer 12

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

Question 13

5 subunits of RNA polymerase holoenzyme

Answer 13

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

Question 14

σ^70

Answer 14

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

Question 15

α

Answer 15

Controls frequency of initiation of transcription

Question 16

β and β’

Answer 16

Polymerase NTPs, transcribe DNA

Question 17

Sigma Factors

Answer 17

• 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

Question 18

Different classes of sigma factors

Answer 18

• σ^70 family (RpoD)
• σ^54 family (RpoN)

Question 19

Different groups of σ^70 family

Answer 19

• 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)

Question 20

Domains of σ^70

Answer 20

• 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

Question 21

When are minor sigma factors expressed

Answer 21

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

Question 22

What is the strength of sigma factor binding determined by

Answer 22

The spacing of the -10 and -35 regions

Question 23

RpoH

Answer 23

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

Question 24

What happens to RpoH under heat stress

Answer 24

• 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

Question 25

How is RpoH regulated in E. coli

Answer 25

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

Question 26

RpoH expression during normal growth

Answer 26

- 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

Question 27

Thermosensitive riboswitch

Answer 27

- 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

Question 28

RpoH during heat stress

Answer 28

- 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

Question 29

DnaK/DnaJ/GrpE chaperone complex

Answer 29

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

Question 30

RpoH degradation cycle feedback loop

Answer 30

- 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

Question 31

Where does RpoH maintain protein folding system

Answer 31

Cytoplasm

Question 32

What is responsible for the protein repair response in the periplasm

Answer 32

RpoE

Question 33

RpoE in E. coli

Answer 33

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

Question 34

RpoE under normal growth conditions

Answer 34

- 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

Question 35

RpoE during stress

Answer 35

- 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

Question 36

Effectors that alterations to folding dependent on

Answer 36

- Metabolites - tRNAs - Temperature

Question 37

What does folding of the 5' UTR of mRNA influence

Answer 37

- Continuation of transcription - mRNA half life - Translation