L9 - Control of Gene Expression in DNA

Question 1

Why does cell diversity result from differences in gene expression?

Answer 1

Cells express a fraction of their genes

Cells express different genes in their disease state

Question 2

When do cells change their expression profile?

Answer 2

In response to signals and environmental cues

Question 3

Regulatory transcription factors

Answer 3

Modulate the level of expression of genes

Question 4

DNA binding proteins

Answer 4

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

Question 5

What is Rox1?

Answer 5

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

Question 6

Alignment of genes from closely related species

Answer 6

Helps identify conserve sequences-

• Non-coding stretches of DNA change during evolution
• Coding and regulatory sequences are conserved

Question 7

Helix-turn-helix DNA binding motif

Answer 7

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

Question 8

Zinc finger DNA binding motif

Answer 8

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

Question 9

Leucine zipper DNA binding motif

Answer 9

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

Question 10

Helix-loop-helix DNA binding motif

Answer 10

Is related to the leucine zipper
Loop enable more flexibility
Can again form dimers or heterodimers
o This increases binding strength

Question 11

Transcription factors are modular

Answer 11

DNA binding domain
Protein binding domain
Regulatory domain
Activation domain

Question 12

How to identify DNA binding proteins?

Answer 12

EMSA – electrophoretic mobility shift assay

DNAse I footprinting

Question 13

EMSA protocol

Answer 13

1. Radioactively label one end of the DNA
   - P32 isotope often used
2. Mix with cell extract or purified protein
3. Load and run samples by gel electrophoresis

Question 14

DNAse I footprinting protocol

Answer 14

1. Radioactively label one end of the DNA – probe
2. Mix with cell extract or purified protein
3. Add DNAse to partially digest the DNA
   - This cuts the DNA in one random position
4. Heat sample to destroy the DNAse and release the binding proteins
5. 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

Question 15

What are the transcription factor forms?

Answer 15

Permissive

Specific/regulatory

Question 16

Permissive transcription factors

Answer 16

General transcription factors are necessary for all transcription

Question 17

Where do permissive transcription factors bind?

Answer 17

At the promoter

To transcription complex

Question 18

Specific/regulatory transcription factors

Answer 18

Activators increase transcription of neighbouring genes

Repressors reduce transcription of neighbouring genes

Question 19

Where do specific/regulatory transcription factors bind?`

Answer 19

Anywhere around gene

Don’t bind to DNA directly and bind to regulatory complex

Question 20

How do specific/regulatory transcription factors function?

Answer 20

Interaction with the RNA polymerase complex
Altering acetylation of DNA
Binding to other transcription

Question 21

What is DNA looping?

Answer 21

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

Question 22

What are insulators?

Answer 22

Block regulatory sequences from affecting neighbouring genes.
Block enhancers from acting inappropriately.

Question 23

What are enhances?

Answer 23

Binding site for transcriptional activators.

Promiscuous – they work on any gene

Question 24

Example of a genetic switch

Answer 24

Trypotophan repressor protein represses genes required for tryptophan synthesis.

Question 25

Ways to regulate a transcription factor

Answer 25

• Protein synthesis
• Ligand binding
• Protein phosphorylation
• Addition of a second subunit
• Unmasking
• Stimulation of nuclear entry
• Release form membrane

Question 26

Transcription factors interact synergistically

Answer 26

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

Question 27

4 ways transcription factors regulate transcription of transcription factors

Answer 27

 Positive feedback loop
 Negative feedback loop
 Flip-flop device
 Feed forward loop