Lecture 4: DNA Binding Proteins Flashcards
What is CAP/CRP?
CAP is an activator.
• When glucose levels are low, cAMP levels rise.
• cAMP binds to CAP and activates it. It then binds to the DNA.
• It is an example of direct and indirect readout.
• CAP can almost bend DNA to 180 degrees.
• There is a HTH with the recognition helix binds in the major groove. There is no code. Side chains are directly making contact with bases.
• There is a twofold axis at TGTGA.
• GC and AT 5 bases apart gives a smooth bend.
• Arginine and glutamate bind for direct readout. This is standard.
• The driving force of binding comes from binding to the phosphate backbone. It only happens if the DNA is correctly bent.
• There is a kink in the middle where to base pairs have split apart. This is called a roll. It is TG stacking which is weaker than average.
How does the trp repressor work?
The trp repressor detects amino acid concentration.
• In high levels of trp, trp binds to the repressor and activates it. Trp biosynthesis therefore stops.
• When trp repressor is bound, the reading heads are held apart at about 34 Angstrom spacing and well ordered.
• When it isn’t bound, the heads collapse inwards and become less ordered.
• It uses a tightly bound water molecule for binding. Blurs the line between direct and indirect readout. Definitely not as accurate as direct readout.
• Either side of the α helix you have a phosphate to have it locked into position.
How do homeodomains work?
Homeodomains are used in development in higher organisms. It’s part of a larger protein.
• You can generate cooperativity.
• The homeodomain recognises 6 bases, around TATA.
• It has 3 helix-turn-helix motifs.
• The recognition helix is in a different position in the groove.
• Direct interaction with asparagine 51.
• Isoleucine 47 also interacts using its methyl group.
• Can’t say much about indirect readout, that is part of the rest of the protein.
How do modular transcription factors work?
You can form homodimers or heterodimers.
• The combinations depend on sequence.
• Examples are Fos and Jun or Myc and Max.
• Larger number of TFs can group together int complexes using modular DNA-binding domains. There may be more than one in a single protein.
• Recognition sequences are often quite short but combinatorial effect allows specificity and builds complexity.
How do zinc finger sequences work?
Zinc finger domains were discovered in TF IIIA and correctly predicted to bind zinc.
• The zinc is used to keep the 30 amino acids in a rigid structure as it can’t fold itself.
• The motif uses cys (C) and his (H)
• CCHH pattern of co-ordinating residues is the most common.
• There are other patterns such as CCCC.
• TFIIIA is present in very large numbers in the genomes of higher eukaryotes.
How can we design zinc fingers?
We can string zinc fingers together, but it is very difficult.
• Each finger recognises 3 bases in a very similar way.
• We have a modular system.
• We use pre-made zinc finger phage display libraries.
• The protein of interest is expressed on the outside of the phage. Part of it is randomised and mutated. We then test the protein binding by testing (e.g. by a column).
• We make two different binding sections and then recombine them. We make 1 and a half fingers which bind to 5 nucleotides.
• We then stitch the proteins together to create 3 fingers which will bind to 9 base pair sequence.
• We can even use this in the clinic.
• We can add zinc fingers to endonucleases. The endonucleases make double stranded breaks. We can cut out a gene or we add the gene of interest.
How does the TATA binding protein work?
TATA binding protein is the most common example.
• It recognises the TATA box for RNA pol II or TFs. It is a core subunit of TFIID.
• It looks highly symmetrical, but it isn’t. It’s all one chain.
• The β chain is highly extended.
• It forces the minor groove out and flattens it.
• It also induces a massive bend.
• It has two kinks, at the first and last step.
• The bend is very strong. It takes advantage of the weak TATA sequence.
• DNA is slightly unwound as well.
• The TATA isn’t recognised by direct readout (it’s in the minor groove).
• There are 5 hydrogen bonds which go to acceptors in the minor groove. They’re identical regardless of being GC or AT.
• The kink has a severe roll of about 40 degrees. It’s similar to CAP except it’s inserting a Phe into the gap.
• If you bend the DNA then you can bring different parts of the DNA together and create protein-protein interactions.
What is integration host factor?
IHF is involved in structuring bacterial DNA.
• It is E. coli’s version of the nucleosome.
• It bends DNA.
• There is some sequence recognition.
• It induces a bend of around 160 degrees.
• It’s quite a small structure and it uses antiparallel β structure.
• It creates a dramatic kink.
• Proline 65 stabilises the kink and it pushes the base pairs apart.
• It is used in the Sin recombinase complex to cleave the DNA.
How are binding proteins involved in DNA repair?
- Uracil Glycosylase (UDG) repairs deamination of cytosine.
- A UG base pair can be recognised by flexibility.
- It scans DNA and squeezes the U out from the base pairing.
- The ribose points outwards.
- The base can then be cut out.
- The protein puts a leucine in to stabilise base stacking. This is often used to perform DNA chemistry (base flipping).
- It only recognises the softness of the base pair.
How does methylation occur?
- Hha1 is a restriction methylase that protects DNA from cleavage through methylation.
- The backbone is pulled out and the bases are flipped out completely.
- It flips out the cytosine and methylates it.
- The protein puts a glutamine side chain in, in order to stabilise base stacking. It forms lots of hydrogen bonds.
- The methyl group comes from a SAM cofactor and at becomes SAH after donation.
- There is lots of direct read out. It is very specific.
- There are phosphate helix interactions.
- There are two anti-parallel β strands in the major groove.
What are nucleosomes?
DNA is packed in nucleosomes.
• Each histone has two turns of DNA wrapped around it.
• Compactness.
• DNA has preferred positions depending on sequence characteristics. .
• Chicken and yeast experiment.
• 1000 times advantage in some positions over others.
• Indirect read out.
• GC rich regions are more bendable towards the major groove.
• AT rich regions tend to be more bendable towards the minor groove.
• If you space these sequences, then the bend will stay in the same direction.
• Because the code is degenerate, you can keep this AT and GC spacing, while retaining the correct code for a gene.
• We can plot roll angles and see and alternating pattern. There is a period of 10 nucleotides. Peaks correspond to kinks.
