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Molecular Biology II Eukaryotic Gene-Specific Transcription Factors

Lecture 20[Page 1]

Additional factors are required to gain more control over the stimulation of transcription than basal transcription alone

In addition to promoters, eukaryotic genes contain large regions that act as binding sites for gene-specific TFs

The sequence-specific binding of gene-specific TFs allows genes to achieve and maintain controlled levels of tissue-

specific expression patterns

Gene-Specific Transcription Factors:

Usually transcriptional activators

Repressors are frequently found in bacterial systems, but a lot less often in eukaryotes as a chromatin packaged

genome is already in a repressed state

Many gene-specific TFs are only expressed in particular cell types

Work together to regulate the expression of a particular set of genes

Gene-specific TFs must be able to specifically bind a subset of genes through sequence-specific DNA-binding

domains. The also need to modulate the activity of promoter bound transcription machinery (RNAP + basal factors)

e.g. stimulate TFIIH to increase helicase activity.

Gene-specific TFs consist of a DNA binding domain (that is specifically folded for DNA binding) and a number of

activation domains.

DNA footprinting

1. DNA is end-labelled

2. DNA is treated with restriction enzymes

3. DNA is denatured to remove protein

4. Fragments are separated using PAGE

5. Bands are visualised by exposing gel to X-ray film

Allows mapping of TF binding sites

Physical Chemistry of Transcription:

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Electrostatic bonds Hydrogen bonds Van derWall forces Hydrophobic interactions

Positive charges in proteins (Arg/Lys/His) will interact with the negativelycharged phosphates in the backbone of

nucleic acids. The shape of a protein is important as well as charge.

Electrostatic interactions between proteins and the DNA backbone provide stability, but no specificity.

Bases are accessible in dsDNA withoutmelting via the major (12 wide) /minor (6) groove

Points of

recognition via the major /minor grooves

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Molecular BiologyII Eukaryotic Gene-Specific Transcription Factors

Lecture 20[Page 3]

Absolute

recognition of the 4 bases is possible via the major groove

TFs that bind the minor groove can onlydistinguish between A-T / T-A from G-C / C-G

DNA-Binding Domains:

All use the -helix in the major groovemethod

I) Helix-turn-helixmotif (manyvariants)

Used in manybacterial TFs (e.g. lac repressor, CAP protein) Also found in manyimportant eukaryotic TFs Contain 2 -helices separated bya loop

o C-terminal helix (recognition helix) fits in major grooveo N-terminal helix stabilises structure and positions recognition helix in groove

Q (glutamine) residues in the recognition helixmake sequence specific contact HTHmotifs are often found as dimers, and so recognise palindromic sequences

o e.g. phage repressorII) Helix-loop-helixmotif

Several TFs involving cell proliferation contain HLHDNA binding domains The c-jun/c-fos heterodimer is a transcriptional activator known as AP-1 Both c-jun & c-fos have been identified as oncogenes

o Can turn a normal cell into a cancerous one ifmutation / overexpression occurs HLHmotifs must dimerise before theycan bind to DNA

III) Zinc-fingermotifs

Contain a zinc atom that is co-ordinated byCys/His residues in a particular configuration Onlyfound in eukaryotes Many1000s proteins in humans are known to contain them The single helix goes into the major groove Usuallywork in tandem Each zinc-finger identifies a 3 nucleotide motif (1/64 triplets)

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Molecular Biology II Eukaryotic Gene-Specific Transcription Factors

Lecture 20[Page 4]

Complex DNA sequences can be recognised by arranging different zinc-fingers in tandem 3 zinc fingers provide a 9 nucleotide recognition sequence

o Useful for making artificial TFs

p53

A tumour suppressor, which, in response to DNA damage slows progression through the cell cycle and initiates

apoptosis if damage is severe

Tumour-specific point mutations occur in many human cancers, of which around 50% contain a p53 mutation

p53 is very complex, so its easy for something to go wrong

p53 acts as a TF and has a central domain which binds DNA in a sequence specific manner

This domain has no structural similarity to any other DNA binding protein

Most of the p53 mutations that bring about cancer are found in the DNA binding domain

Activation Domains:

Once a target site has been recognised by gene-specific TFs, the basal transcription machinery has to be stimulated,

which is carried out by activation domains.

Many are unstructured which means they cannot be crystallised. This makes analysis harder.

Can help to create open chromatin domains