8 - Intro to Bacterial Gene Regulation Flashcards
Central dogma
DNA –Transcription–> RNA –Translation–> Protein
Gene naming convention
lower case, italics (e,g, rpoH)
Protein naming convention
Uppercase, no italics (RpoH)
Outcomes of transcription
tRNA, mRNA, rRNA
Regulon
Multiple genes in different locations controlled by the same type of promoter thus resulting co-ordinated expression
Operon
Multiple genes in the same location, controlled by a single promoter
Gene
Entire nucleic acid sequence necessary for expression of a gene product
Why do bacteria regulate gene expression
To express a subset of proteins to permit the bacterium to survive current conditions
Examples of global responses
- SOS response (sudden global DNA damage)
- Starvation response
- Heat stress response
Specific responses
- lac operon to utilise lactose as an energy source
- trp operon to synthesise tryptophan
Hierarchical control mechanisms for gene expression
- 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)
Mechanisms controlling gene expression at the transcriptional level
- Different promoters bind different sigma factors of RNA polymerase
- DNA binding proteins bind the promoter region acting as repressors or activators of transcription
5 subunits of RNA polymerase holoenzyme
- 2 large subunits β and β’
- 2 copies of smaller α
- 1 copy of σ^70
σ^70
- Initiation factor
- Interacts with promoter
- Binds to specific sequences near -10 and -35 boxes
α
Controls frequency of initiation of transcription
β and β’
Polymerase NTPs, transcribe DNA
Sigma Factors
- 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
Different classes of sigma factors
- σ^70 family (RpoD)
- σ^54 family (RpoN)
Different groups of σ^70 family
- 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)
Domains of σ^70
- 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
When are minor sigma factors expressed
In response to physiological signals such as starvation, temperature, growth phase
What is the strength of sigma factor binding determined by
The spacing of the -10 and -35 regions
RpoH
- Heat stress proteins have RpoH promoter sequences
- Under normal conditions, RpoH expression is low therefore, the expression of RpoH controlled regulon is low
What happens to RpoH under heat stress
- 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
How is RpoH regulated in E. coli
- Post-translational level (sequestration and
degradation) - Post-transcriptional (mRNA structure prevents translation)
- Transcriptional level (promoter recognised by different sigma factors for induced expression)
RpoH expression during normal growth
- 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
Thermosensitive riboswitch
- 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
RpoH during heat stress
- 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
DnaK/DnaJ/GrpE chaperone complex
- Preferentially recognizes misfolded proteins in the cytoplasm
- Associates with ClpB foldase that re-folds proteins to correct conformation
RpoH degradation cycle feedback loop
- 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
Where does RpoH maintain protein folding system
Cytoplasm
What is responsible for the protein repair response in the periplasm
RpoE
RpoE in E. coli
- Responds to misfolded proteins in the periplasm
- Gram negative bacteria
- Must transmit response signal from periplasm to cytoplasm compartments across the periplasmic membrane
RpoE under normal growth conditions
- 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
RpoE during stress
- 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
Effectors that alterations to folding dependent on
- Metabolites
- tRNAs
- Temperature
What does folding of the 5’ UTR of mRNA influence
- Continuation of transcription
- mRNA half life
- Translation
