L15 : Regulation of Gene Expression in Prokaryotes

Question 1

Why is gene expression regulation essential in bacteria?

Answer 1

Adaptation to environmental changes and avoid constitutive protein expression, which would be energetically inefficient

Question 2

Why is multi-layered control necessary?

Answer 2

To integrate multiple systems and allow rapid adaptation to environmental changes

Question 3

What are the 3 main levels of gene expression regulation?

Answer 3

Transcriptional control
- initiation
Post-transcriptional control
- elongation, termination, translation
Post-translational control
- modifications, proteolysis

Question 4

What is the role of sigma factors? Example

Answer 4

Recognise specific promoter elements
Eg. -10 and -35 elements of core promoter

Question 5

What is sigma factor competition?

Answer 5

Different sigma factors with varying affinity compete for binding RNAP
Affects which genes are transcribed

Question 6

What do TFs do in bacteria?

Answer 6

Can recruit RNAP directly or can compensate for poor -35 element

Question 7

How does repression of transcription occur?

Answer 7

Repressors block RNAP from accessing the promoter or elongating the transcript

Question 8

How can repressors be regulated?

Answer 8

Repressors can be released in the presence of certain ligands

Question 9

Does RNAP move unformaly during elongation?

Answer 9

No
Can pause at specific DNA sequences
Pausing sites are inherent to RNAP and template

Question 10

What causes transcription termination in bacteria?

Answer 10

Intrinsic terminators or Rho-dependent terminators

Question 11

What is an intrinsic terminator? Where is it common?

Answer 11

Easily recognised
Common in E.coli and B.subtilis
Rare in M.tb

Question 12

What is a Rho-dependent terminator? Where is it common?

Answer 12

Difficult to recognise
Common in E.coli and M.tb
Rare in B.subtilis

Question 13

What is conditional termination?

Answer 13

Regulatory process where transcription may terminate or continue based on environmental conditions

Question 14

How does M.tb typically infect humans?

Answer 14

Via inhalation of droplets, infecting macrophages in lungs

Question 15

What is dormancy in M.tb

Answer 15

Non-replicating persstent state associated with latent infection

Question 16

What stressors induce dormancy in M.tb

Answer 16

Hypoxia
Starvation
NO
CO
Low pH

Question 17

What is a granuloma?

Answer 17

Structure formed by immune system to contain M.tb infection

Question 18

How does M.tb survive in granulomas?

Answer 18

Adapting to stress, including hypoxia and nutrient limitation

Question 19

What is DosR?

Answer 19

Transcription factor and master regulator of dormancy in M.tb

Question 20

What induces DosR activation?

Answer 20

Hypoxia
NO
CO
Low pH
Intracellular macrophage environment

Question 21

What type of system is DosR part of?

Answer 21

Two component regulatory system
With DosS and DosT kinases

Question 22

Where does DosR bind?

Answer 22

To multiple sites upstream of TSSs, often overlapping in -35 region

Question 23

What does DosR regulon control?

Answer 23

Over 50 genes
Regulon associated with alternative metabolism and unversal stress response

Question 24

What is a two component system?

Answer 24

Signal transduction system with sensor kinase and response regulator

Question 25

What is autophosphorylation?

Answer 25

Process where kinase phosphorylates itself using ATP

Question 26

How is the response regulator activated?

Answer 26

Sensor kinase undergoes autophosphorylation Transfer of phosphate onto response regulator

Question 27

What is the difference between two component and phosphorelay system?

Answer 27

Phosphorelay system involves multiple phosphorylation steps before response regulator is activated

Question 28

What are the 3 main experimental approaches to define the DosR regulon?

Answer 28

1. Overexpression fo DosR 2. ChIP seq to identify DNA binding sites 3. Transcriptomics to measure gene expression changes Assessment of binding events with transcriptional regulation

Question 29

What is the purpose of overexpressing a TF like DosR?

Answer 29

Study regulon and observe downstream gene expression changes

Question 30

How is DosR overexpressed? Problem?

Answer 30

Using an inducible promoter system, allowing controlled expression of DosR May cause off-target binding

Question 31

How does ChIP seq work?

Answer 31

1. Crosslink TFs to DNA using formaldehyde 2. Immunoprecipitate the complex using FLAG tag 3. Sequence the bound DNA

Question 32

What does ChIP seq tell you?

Answer 32

Which DNA region a TF physically binds to across genome

Question 33

What is transcriptomics used for?

Answer 33

eg. microarray Profiling gene expression across genome

Question 34

What are tiled microarrays?

Answer 34

Arrays with overlapping probes that cover entire genome Used for detecting changes in expression

Question 35

What are limitations fo tiled microarrays?

Answer 35

Lower resolution Do not give nucleotide level precision Compared to other transcriptomics methods (RNA-seq)

Question 36

Why combine ChIP-seq with transcriptomics in DosR research?

Answer 36

To link TF binding events with changes in gene expression Distinguish direct targets (bound and regulated by DosR) from indirect effects

Question 37

How are PPIs between DosR and SigA investigated?

Answer 37

GST pulldown assay - DosR fused to GST and incubated with bacterial lysate - Pull down using glutathione agarose beads - Interacting proteins identified - Detect SigA with anti-His antibodies

Question 38

What was the significance of mutating surface charged residues on DosR?

Answer 38

Mutations abolished DosR-SigA interactions Indicate electrostatic interactions critical for binding

Question 39

What did investigation of DosA-SigA interactions under different conditions show?

Answer 39

Critical for survival during hypoxia Not required during aerated growth

Question 40

How does DosR activate transcription with weak -35 element?

Answer 40

By interacting with SigA to compensate for poor promoter sequences