Gene regulation (Dr Curran) Flashcards
How do organisms become complex?
-Complexity arises from modularity: • 4 BASES • 20 AMINO-ACIDS • 1000 PROTEIN FOLDS -many combinations-more complex • DIFFERENTIAL PROTEIN-PROTEIN INTERACTIONS. • DIFFERENTIAL PROTEIN-DNA INTERACTIONS. • DIFFERENTIAL PROTEIN-RNA INTERACTIONS
What is the most important component in a multicellular organism?
-cell to cell interaction
What is Data?
A representation of facts, concepts, or instructions in a formalized manner suitable for: • COMMUNICATION. • INTERPRETATION. or • PROCESSING. by humans or by automated means.
What is information?
Information is the result of: • PROCESSING. • MANIPULATING. and • ORGANISING Data
What is the significance of mice DNA being very similar to human?
- THE OVERALL GENE SEQUENCES ARE THE SAME.
- IT IS HOW THEY ARE REGULATED THAT IS DIFFERENT
How can we effect global changes in prokaryotic gene expression?
-DNA polymerase creates RNA strand, this begins at promoter
-RNA polymerase initiated by sigma factor
-Change the RNA polymerase sigma factor.
-Changing the RNA polymerase’s Sigma factor directs the Polymerase to different promoters:
• Normal sigma factor (σ70) binds to RNA polymerase and recognizes sequence in promoter to initiate transcription
• Heat shock disables σ70
• Product of rpoH gene, σ32 binds to sequence in promoter of heat shock genes when heat stressed and starts transcription
-simply change sigma factor to regulate gene expression
-‘global’ because it affects lots of cells, lots of beings
How can we effect local changes in prokaryotic gene expression?
• REPRESSOR PROTEIN(S) BIND TO DNA AND INHIBIT TRANSCRIPTION. -THIS IS NEGATIVE CONTROL • ACTIVATOR PROTEIN(S) BIND TO DNA AND ENHANCE TRANSCRIPTION. -THIS IS POSITIVE CONTROL \+local because it affects a single gene
What are the types of genes on an individual gene level?
• Regulated genes: The level of the encoded protein varies.
– Inducible genes: Off until required.
– Repressible genes: On until not required.
• Constitutive genes : The level of the encoded protein is constant.
What is an Operon and what is an Operator?
- Operon: Two or more genes co-ordinately regulated by a single promoter and terminator.
- Operator: Section of DNA that binds a regulatory protein in an operon
How is catabolism regulated? Use an example to explain.
• Enzymes break down molecules for energy generation. (catabolism)
• Their presence in the cell is only required when the molecules are present.
• E.g. Lactose Metabolism – Breaking down
lactose to generate ATP:
-Complementation Analysis of mutants identifies
lactose utilization genes
- Jacob, Monod et al. isolated many Lac- mutants unable to utilize lactose (mutant cant make lactose)
- Complementation analysis identified three genes
(lacZ, lacY, and lacA) in a tightly linked cluster (all three enes cant make lactose)
-Experimental evidence for repressor protein
- Isolated mutant in lacI gene (not boud to other ones)
– Constitutive mutant – synthesized β- galactosidase and lac permease even in absence of lactose (inducer)
– lacI must be a repressor – cells must need lacI protein product to prevent expression of lacY and lacZ in absence of inducer
-Inducer releases repressor to trigger enzyme synthesis
- Addition of lactose inducer caused β – galactosidase synthesis to be induced.
- Conclusion: Inducer binds to repressor so repressor can not bind to DNA
- Allosteric effect - inducer bound to repressor protein changes conformation so it can not bind to the promoter in DNA
What is the Operon theory of gene regulation?
-Operon Theory of gene regulation
- Jacques Monod and Francois Jacob Pasteur Institute in Paris
-one protein regulating production of other protein
-(a) Lactose absent, repressor active, operon off
-(b) Lactose present, repressor inactive, operon on
-(a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesised
-(b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized
-Repressor has binding domains for operator and for the inducer
-Positive control increases transcription of lacZ, lacY, and lacA:
• cAMP binds to CRP (cAMP receptor protein) when glucose is low
• CRP binds to regulatory region
• Enhances activity of RNA polymerase at lac promoter -Changes in the operator can also affect repressor activity
Summarise the level of transcription based on glucose and lactose levels.
- High Glucose and High Lactose (low level transcription)
- High Glucose and No Lactose (no transcription)
- Low Glucose and High Lactose (high level transcription)
- Low Glucose and No Lactose (no transcription)
What is trans effect?
Genes are regulated even when two DNA segments are NOT physically adjacent.
What is Cis-acting element?
A DNA segment that must be adjacent to the gene(s) that it regulates.
Describe the experiment they used to showcase the operon.
- recipient cell has LacI mutation, operon turned on, not making repressor, unable to regulate operon (break cells open and measure LacZ to check, turns yellow if it has)
- donor cell mutated as well however has a plasmid with repressor, correctly working, has LacZ
- mix them and plasmid fuses and switches off mutation,makes enough repressor to bind to both operators, regulating pathway normally, lactose is taken up
Do proteins and DNA act in cis or trans?
-Proteins act in trans
-DNA sites act only in cis
• Trans acting elements can diffuse through cytoplasm and act at target DNA sites on any DNA molecule in cell
• Cis acting elements can only influence expression of adjacent genes on same DNA molecule
Describe a defective regulation due to a trans mutation
- Inducible synthesis is restored by the F’ plasmid.
- lacI+ gene encodes a diffusible element that acts in trans by binding to any operator it encounters regardless of chromosomal location lacI on the F’ plasmid encodes a supperrepressor which cannot bind inducer.
- All operator sites (O+) eventually occupied by superrepressor.
- lacIS encodes a diffusible element that acts in trans by binding to any operator regardless of chromosomal location
Describe a defective regulation due to a cis mutation
- Presence of O+ sequence on F’ plasmid does not compensate for Oc mutation on bacterial chromosome.
- Operator is cis acting element
