DNA-protein interactions L1-3

Question 1

How can DNA binding proteins recognise DNA?

Answer 1

Particular sequences (e.g. Transcription factors)
Particular DNA structures (e.g. some proteins involved in recombination) Cruciforms/flaps
Particular types of damaged DNA (repair proteins)

Histones appear to bind with little specificity but high affinity

Question 2

What is SUPERSHIFT?

Answer 2

Confirming identity of a DNA binding protein that binds to site of interest
If you don’t know the protein, instead of mixing DNA with purified protein mix with cell extract (mixture of proteins)-observe shifts
Add antibody to protein X-if it successfully binds it produces an even larger complex and the band will run in a different position. If no effect observed an alternative protein must be bound since it isn’t complementary to the existing antibody

Question 3

What charge is DNA?

Answer 3

Negative

Every bp carries TWO negative charges

Question 4

Van der waals interactions

Answer 4

The majority of interactions b/w DNA and protein are van der waals-these interactions are individually weak and add little specificity but overall make a considerable contribution to the affinity of the interaction

Question 5

What base is in hydrophobic interactions

Answer 5

Thymine, has a CH3 group which can interact with non polar amino acid side chains to minimise interactions with water-form clusters

Question 6

Hydrogen bonds

Answer 6

Dictate the specificity of most protein-DNA interactions

DNA contains multiple H bond donors and acceptors

Question 7

Is the majority of recognition done in the major or minor groove?

Answer 7

MAJOR
More bases visible (4)
More accessible
More information content -4 bases are not symmetrical so can distinguish left/right orientation of bases
The minor groove only consists of 3 bases and is often symmetrical so can only distinguish b/w two possibilities

Question 8

MAJOR/MINOR groove

Answer 8

In the major groove each base contributes 2 bits of info
In the minor groove the purines contribute 2 whilst the pyrimidines only contribute 1

AMIDE groups=typical donors
CARBOXYL groups=typical acceptors

Question 9

What base does DAM attach a methyl group to?

Answer 9

N6-methyl adenine
This can change what the protein looks for in the sequence of DNA. It changes the substrate and interferes to allow or disallow binding

Question 10

What amino acid does guanine regularly bind to via H bonds?

Answer 10

Arginine
Arginine has 2 H bond donors and Guanine has 2 H bond acceptors
2 part interaction

NB/ Arg residues can interact with DNA in a variety of orientations, recognising a variety of bases

Question 11

What interactions are important for the Trp repressor?

Answer 11

Water mediated H bonds are the principle determinants for sequence-specific DNA binding to the Trp repressor.
In other cases water-mediated H bonds also contribute significantly to the sequence-specificity of the protein-DNA interaction.

Question 12

What is the most widely used secondary structure to bind to DNA?

NB/ Secondary structures (alphahelices/betasheets) are held together by H bonds in the BACKBONE of the polypeptide not the side chains. Laying this structure in the major groove doesn’t effect the backbone H bonds. Side chains are available to make interactions with the sides of bases in the major groove

Answer 12

The alpha helix. (Likely to be part of a helix-turn-helix motif in a super secondary structure)
The width and depth of the MAJOR groove are a close match to the dimensions of an alpha helix.
When the alpha helix binds to the negative DNA the electronic distribution ends up being skewed-produces a dipole moment (N=delta positive, C=delta negative). Laying the alpha helix in the right place the slight positive charge can make appropriate connections with the negatively charged DNA backbone. (Increases affinity and helps helix to align in the right place)
Common protein motifs that proteins use in order to arrange an array of amino acid side chains to read longer DNA sequences
An atypical example is the bacterial met repressor protein which binds to the major groove in DNA via a double anti-parallel beta strand

Question 13

In a helix-turn-helix motif what helix is the recognition helix?

Answer 13

The most C terminal one.
Interactions b/w helices maintain the structure. The other helix helps to stabilise the recognition helix.
Homeotic mutations such as fly legs for antennae are mutations in a helix turn helix structure of a transcription factor
Homeodomain proteins are a specialist form of helix-turn-helix.
HTH proteins are often DIMERIC and the physical distance between the recognition helix of the monomers are 3.4nm which is the length of 10bps in DNA.

Question 14

Why in a dimeric protein with 2 HTH motifs is the spacing of these 10bps apart?

Answer 14

The protein can only interact with the DNA from one side, it sits and binds on one surface.
DNA bases rotate around 36 degrees-10bases go around 1 helix

Question 15

Why dimerise the proteins?

Answer 15

To bind only a small number of locations within a large genome.

Question 16

How many bases does a single alpha helix read?

Answer 16

Since the alpha helix lays at a tangent to DNA it normally only read 5bases on the top half yet if it has a few loops etc on the end it provides flexibility the most that a single alpha helix can read is SIX base pairs!

Question 17

leucine zipper proteins

Answer 17

Eg Fos/Jun transcription factor
Long alpha helices 60aa long
Join together via leucine residues at C terminal ends
Proteins clip over DNA in two major grooves
Depending on seq of zipper protein you can alter specificity of the protein
Leucine zipper proteins turn genes ON when they bind next to the gene. Changing the patterns of these proteins dictates which site/gene it binds to

Question 18

Zinc fingers

Answer 18

Proteins which contain zinc fingers increases the specificity and affinity of DNA
Most natural zinc fingers will have 6,7 or 9 and read a seq of 18-20bps long. (extend the recognition seq)
A single zinc finger motif contains a 2 stranded beta sheet and 1 alpha helix
A zinc is chelated to CYSTEINES acts like a pin to hold the protein together and is not involved in interacting with DNA. Alpha helix interacts with the DNA.
Each zinc finger reads a THREE base pair segment.
There is a sufficient code that you can build a zinc finger on to recognise any 3base seq that you want.
Can use Zinc fingers or CRISPR to target proteins and produce a ds break. (attach nuclease to artificial zinc finger)

Question 19

How long are TALES in nature?

Transcription activator like effectors

Answer 19

1.5-33.5 tandemly repeated DNA binding motifs ( TAL repeats)

Question 20

How long is each TAL repeat in terms of amino acids?

Answer 20

34aa and consists of 2 helices joined by a linker
Residues 12 and 13 in the linker are HYPERVARIABLE and define the DNA specificity in a predictable way

1.5 and 33.5 repeats that are usually 34 residues in length

Question 21

How do TALEs bind to DNA?

Answer 21

The linker (unstructured region) interacts with the DNA rather than the helices.
Structure of a 12 repeat TALE bound to DNA forms a right handed superhelical structure and tracks the major groove of the DNA double helix. Each structure/domain sits in the major groove and follows this groove around behind the DNA and out the front again. Loops stick out from middle of 12 helical flower structure and these are where the 12/13 hyper variable loops are.
Each TAL repeat (34aa long) reads only a single base (whilst h-t-h motifs read a whole range of bases

Question 22

TALES

Answer 22

TALEs are secreted by some bacteria when they infect plant cells. As they invade the plant cells they overtake the genetic programming of those plant cells so that the plants do what the bacteria want. They inject these transcription factors/regulatory proteins into the plant cells. These will bind to the plant promoter sequences and change the levels of expression and gene proteins

Question 23

Which residue in the 34aa TAL specifically interacts with a single base?

Answer 23

residue 13
with this code you are able to design proteins that will read any seq you want
Artificial nucleases incorporating designed arrays of TAL repeats fused to nuclease domain (TALENS) can be used for genome engineering (similar to CRISPR) can also be done with zinc fingers
TAL repeats are used as a targeting domain that will bind to specific regions in the genome that you want to be cut. Can recognise a site up to 24 bases
TALENS USE RNA TO BIND TO DNA

Question 24

Name three proteins that make contacts with the minor groove

Answer 24

Lac repressor (dimer) - binds 2 extra helices in the minor groove in addition to the h-t-h recognition helix in the major groove.

TBP (tata binding protein/psuedodimer-monomeric protein with 2 more or less identical repeats)- The DNA is bent almost back on itself and residues interact with the minor groove

Homeodomain proteins-minor groove is too narrow for alpha helix yet binding proteins like these use loops to interact with the minor groove

Other proteins distort the DNA structure so that larger structures can be inserted into the minor groove.
Proteins achieve increased binding energy and/or specificity by binding to the minor groove in addition to the major groove using an unstructured/floppy loop

Question 25

What are indirect readouts?

Answer 25

Where DNA-binding proteins show specificity for a particular NT seq even though NO CONTACTS are made with the bases concerned.
No specific hydrogen bonds made to bases yet it is still able to bind more tightly to some sequences than others

Question 26

A sequence that causes a bend in the DNA

Answer 26

5’-AAAAnnn-3’ causes a slight bend/gentle curve in the DNA and is preferentially bound by some proteins egHNS

Question 27

How many bps of DNA is associated with a single nucleosome core

Answer 27

About 200bps
Proteins that bend or distort DNA when they bind often show a particular preference for certain NT seqs because they are easier to bend/distort