INDM 3007. Lecture 9. Proteins interact with DNA: how do they know where to bind?. DNA appears to a homogenous molecule, no specific features to recognise is this true?. What is unique to a particular stretch of DNA? Local shape of DNA Nucleotide sequence.
DNA appears to a homogenous molecule, no specific features to recognise is this true?
What is unique to a particular stretch of DNA?
Local shape of DNA
DNA binding proteins use these two features to recognise a particular sequence
The morphology of DNA is dependent on the DNA sequence. Some sequences introduce bends in DNA for example
These structural features are recognised by proteins, much like in the ‘lock and key model’ for enzymes
Local DNA structure
DNA is not a straight tube
The DNA helix has two ‘grooves’: sequences introduce bends in DNA for example
the major groove
the minor groove
to which proteins bind
The nucleotide bases are on the inside of the helix
DNA binding proteins do not open the helix, so what do they recognise?
DNA binding proteins ‘see’ the edges of the basepairs in the major or minor groove
The protein ‘sees’ a particular array of these, which is different for each of the four base pairs
Note that the edge pattern for G:C is different than the one for C:G
What is it that these proteins interact with:
Hydrogen bond donors
Hydrogen bond acceptors
Notice that in the major groove, every base pair has a unique pattern, wherease the minor groove only has two distinct patterns.
The major groove is therefore more informative than the minor groove
Based on structure comparisons it turned out that many bacterial DNA binding proteins contain a conserved domain of two alpha helices: helix-turn-helix motif
What parts of the protein are involved in DNA recognition
Mutations in helix 2 prevent DNA binding, which can be suppressed by mutations in the DNA sequence of the operator
Swapping helix 2 between two different repressors also swapped the operator to which the proteins bind
This shows that helix 2 is involved in DNA recognition
Using X-Ray crystallography to determine the 3-D structures of proteins bound to DNA, the protein domains binding to DNA were revealed
Helix 2 inserts into the major groove of DNA, whereas helix 1 lies across the groove
Helix 2 interacts with the base pair edges
Helix 1 contacts the sugar phosphate backbone
How does this work?
Specific amino acids, on the side of the helix facing DNA, interact with the base pair edges through hydrogen bonding
The number of interactions between helix 2 and the DNA sequence determines the strenghth of DNA binding.
Helix turn helix motif of Cro
repressor protein (phage Lambda)
Interaction between Cro and DNA
Explains why the same protein can bind to different, yet related sequences with different affinities. We saw this for the LysR type proteins!
helix interact with the base pair edges through hydrogen bonding
Many DNA binding proteins are dimers, e.g, the LysR type proteins and CAP
This means that there are two helix-turn-helix motifs
per dimeric protein
These will interact with two adjacent major grooves, ie 10 bp apart
(CbbR binding site, lecture 5)
The DNA recognition site is therefore frequently an inverted repeat
The helix-turn-helix motif is often employed by bacterial DNA binding proteins, but there are other motifs.
Will be discussed by Patrick Caffrey
Non specific DNA binding proteins do not have sequence specific interactions with DNA. Histones for example rely on electrostatic interactions:
DNA phosphate back bone is negatively charged
Protein is positively charged.
It allows regulators binding far upstream (see lecture 5) to contact the RNA polymerase
Integration host factor, IHF, is another example of proteins that do not make use of a HTH motif to bind to DNA
This protein induces an 180o bend in the DNA
Sigma54 RNA pol