L9 - Control of Gene Expression in DNA Flashcards
Why does cell diversity result from differences in gene expression?
Cells express a fraction of their genes
Cells express different genes in their disease state
When do cells change their expression profile?
In response to signals and environmental cues
Regulatory transcription factors
Modulate the level of expression of genes
DNA binding proteins
Are + charged
- Allows the protein to stick to the - charged phosphate backbone
Reach into the major groove
- H bonds form in the major groove
Binding site - response element
- Transcription factors recognise these short stretches of DNA through interactions with base pairs
What is Rox1?
Binds to 8 sites in 3 yeast genes
- Hem13
- ANB1
- ROX1
Different sites have different affinity for the protein
More genes can be identified once a consensus sequence has been found
Alignment of genes from closely related species
Helps identify conserve sequences-
- Non-coding stretches of DNA change during evolution
- Coding and regulatory sequences are conserved
Helix-turn-helix DNA binding motif
Recognition helix inserts into major groove
Helix-turn-helix proteins bind as dimers to two consecutive major grooves
The DNA recognition sequence is palindromic – mirror image of other sequence
Zinc finger DNA binding motif
Four amino acids hold the zinc atom in place
Alpha helix recognises two bases and interact with major groove
Can have multiple zinc finger motifs in a row
Use arginine and histamine to interact with bases and form H bonds
Leucine zipper DNA binding motif
Alpha helical monomers are held together by hydrophobic amino acids
Can dimerise with themselves or form heterodimers with other leucine zippers
Homodimers bind symmetrical sequences
Heterodimers bind non-identical sequences
Helix-loop-helix DNA binding motif
Is related to the leucine zipper
Loop enable more flexibility
Can again form dimers or heterodimers
o This increases binding strength
Transcription factors are modular
DNA binding domain
Protein binding domain
Regulatory domain
Activation domain
How to identify DNA binding proteins?
EMSA – electrophoretic mobility shift assay
DNAse I footprinting
EMSA protocol
- Radioactively label one end of the DNA
- P32 isotope often used - Mix with cell extract or purified protein
- Load and run samples by gel electrophoresis
DNAse I footprinting protocol
- Radioactively label one end of the DNA – probe
- Mix with cell extract or purified protein
- Add DNAse to partially digest the DNA
- This cuts the DNA in one random position - Heat sample to destroy the DNAse and release the binding proteins
- Run samples by gel electrophoresis
- Can be used to identify where a protein binds
- Ladder effect on the gel
- DNA binding protein protects the bound DNA from cleavage- Blank space on gel
What are the transcription factor forms?
Permissive
Specific/regulatory
Permissive transcription factors
General transcription factors are necessary for all transcription
Where do permissive transcription factors bind?
At the promoter
To transcription complex
Specific/regulatory transcription factors
Activators increase transcription of neighbouring genes
Repressors reduce transcription of neighbouring genes
Where do specific/regulatory transcription factors bind?`
Anywhere around gene
Don’t bind to DNA directly and bind to regulatory complex
How do specific/regulatory transcription factors function?
Interaction with the RNA polymerase complex
Altering acetylation of DNA
Binding to other transcription
What is DNA looping?
Important role in forming DNA/protein complexes
Chromatin does not bend easily so for two proteins to interact they need to bind to
- Directly neighbouring DNA sequences
- Sites that are >500 bp apart
What are insulators?
Block regulatory sequences from affecting neighbouring genes.
Block enhancers from acting inappropriately.
What are enhances?
Binding site for transcriptional activators.
Promiscuous – they work on any gene
Example of a genetic switch
Trypotophan repressor protein represses genes required for tryptophan synthesis.
Ways to regulate a transcription factor
- Protein synthesis
- Ligand binding
- Protein phosphorylation
- Addition of a second subunit
- Unmasking
- Stimulation of nuclear entry
- Release form membrane
Transcription factors interact synergistically
Binding of one transcription factor to another helps prevent them from falling off DNA
Each protein needs to lose two interactions to fall off
Binding of one transcription factor to DNA may enable another transcription factor to bind
4 ways transcription factors regulate transcription of transcription factors
Positive feedback loop
Negative feedback loop
Flip-flop device
Feed forward loop