Contol of gene transcription Flashcards
Changes in gene expression underlie what?
The diversity of cell types in the body
All cells have the same … but different sets of …
Genes
Proteins
What can affect which genes are expressed?
Responses to signals and other cues in the enviroment
Cells also change gene expression in some disease states
What affects the level of translation?
The level of transcription in a cell
High levels of transcription can mean more RNA is produced so more protein is made
What is the charge of DNA binding proteins?
They are positive
What is the target of DNA binding proteins?
The major groove
What is the charge of the phosphate backbone?
Negative
Unlike proteins, DNA has a limited what?
Topology
Regulatory proteins must recognise what on the DNA?
A specific nucleotide sequence
Why does the outisde of the helix contain information?
So that the double helix does not have to open
The edge of each pair is exposed at the surface, presenting a distinctive pattern of what?
H-bond acceptors
H- bond donors
Hydrogen atoms
Methyl groups
Which groove is marked differently for each pair?
The major groove
Give the pattern of the major groove for:
G-> C
A- > T
Know this to know the reverse
G to C
H bond acceptor, H bond acceptor, H bond donor, Hydrogen atom
A to T
H bond acceptor, H bond donor, H bond acceptor Methyl group
Molecular recognition usually relies on what?
Exact fit between the two surfaces of a molecule
How do gene regulatory proteins recognise a specific DNA sequence?
The surface of the protein is complementary to the features of the double helix in that region
Proteins make a series of contacts using which 3 interactions?
Hydrogen bonds
Ionic bonds
Hydrophobic interactions
How are interactions between the proteins and DNA strong?
Even though each contact is weak, there are about 20 contacts making them hard to break
Where does ROX1 bind?
Known to bind to eight sites in three yeast genes
Do all the DNA binding sites have the same affinity for the ROX1 protein?
No
Describe the structure of a helix-turn-helix DNA binding motif
2 Alpha helices connected by a short constitute of amino acids
The 2 alpha helixes are held at a fixed angle
The more C terminal is called the recognition helix as it fits in to the major groove of DNA, making specific contacts
Helix-turn-helix proteins tend to bind as dimers to what?
Two consecutive major grooves
The DNA recognition site for Helix-turn-helix DNA binding motif is what?
Palindromic
Describe the structure of the zinc finger binding domain?
Four amino acids which hold the zinc atom
Held by cis-cis-his-his
Has three fingers
The alpha helix of the zinc finger can recognise how many bases?
Two
Describe the structure of a leucine zipper DNA binding motif
The alpha helical monomers are held together by a hydrophobic amino acid eg leucine
Forms a Y shaped structure allowing the side chains to contact the major groove of DNA
What do homodimers and herterodimers bind?
Homo = symetrical sequences Hetero = Non identical sequences
What is the purpose of heterodimerisation?
It expands the repetoire of DNA sequences that gene regulatory proteins can bind to
The helix-loop helix is related to what?
The leucine zipper
What is the effect of DNA binding proteins binding as dimers?
It increases their strength - also known as syngergy
What are the domains of a transcription factor?
DNA binding domain
Protein binding domain
Regulatory domain
Activation domain
How can DNA binding proteins be identified?
DNAse I footprinting
List the steps in DNAse footprinting
1) Radioactively label one end of the DNA
2) Mix DNA with cell extract
3) Add DNAse to partially digest the DNA
4) Heat the sample to destroy DNAse and release the binding proteins
5) Run samples by gel electrophoresis
This shows regions DNA protected from digestion due to protein binding
DNAse I footprinting can also be used to identify what?
Where a protein binds on a sequence of DNA
What is EMSA?
Electrophoretic mobility shoft assay
What is EMSA used for?
Identify DNA binding proteins
Give the steps in EMSA
1) Radioactively label one end of the DNA
2) Mix with the cell extract (or purified protein)
3) Run samples by gel electrophoresis
Lanes with a protein bound will move through the gel slower
What are the three forms of transcription factors?
Permissive
Specific
Regulatory
What are permissive transcription factors?
General - neccessary for all transcription
Bind at the promoter
How do regulatory transcription factors function?
Interactions with the RNA polymerase complex
Altering acetylation of the DNA
Binding to other transcription factors
Where do regulatory transcription factors bind?
Anywhere around the gene - it can be far away
Or they dont bind DNA directly and bind to a regulatory complex
What does DNA looping play an important role in?
Forming DNA/protein complexes
Because chromatin does not bend easily it is thought that what is needed for two proteins to interact?
They need to bind directly to neighbouring DNA sequences
OR to two sites that are >500 base pairs apart
What is the role of insulators and barriers?
They block regulatory sequences from affecting neighbouring genes
Which inputs can alter gene expression by integrating?
Activating proteins
Inhibition proteins
Genetic switches respond to what
Extrinsic or intrinsic regulation
What is tryptophan repressor protein?
A repressor protein which represses genes required for tryptophan synthesis
List ways that a transcription factor could be regulated
a) Protein synthesis
b) Ligand binding
c) Covalent modification
d) Addition of a second subunit
e) Unmasking
f) Stimulation of nuclear entry via removal of an inhibitory protein allowing it to enter the nucleus
g) Release from membrane
Transcription factors can interact in what way with each other on the DNA?
Give an example
They act syngergistically
eg just A = 1 unit of transcription
Just B = 1 unit of transcription
A + B = 100 units of transcription
Binding of transcription factors on the DNA helps to prevent what and also helps with what?
Prevents them falling off the DNA
Helps as binding of one may enable another to bind
What are the ways in which transcription factors can regulate the transcription of transcription factors?
Positive feedback = more made
Negative feedback = less made
Flip flop device - two inhibit each other
Feedforward loop
