Lecture 1
- Only the edges of the base pairs
are accessible.
- The hydrogen bonding patterns
are different for the two grooves.
- In the minor groove the only
difference between GC and AT is the 2-
amino group of G
Methods for detecting interaction with DNA
1. Bandshifts- no good for weak interactions as the complex dissociates in the gel
- Bandshift experiments can be used to determine the sequence of the DNA- binding site
of a protein complex using a technique known as SELEX (systematic evolution of ligands
by exponential enrichment) This involves binding the protein of interest to a random
DNA sequence contained within a longer oligonucleotide template and separating the
bound and unbound DNA in a bandshift gel. The bound DNA is then amplified. The
consensus binding site is known after multiple amplifications.
- They can also be used to visualize protein- protein interactions between a DNA binding
protein and other non-DNA binding proteins in what is called a supershift assay in this
assay the binding of a second protein to a protein DNA complex is visualized by a
further retardation of mobility.
Examples is the use of antibodies to confirm the identity of a protein in a protein- DNA
complex as in the identification of a basic helix-loop-helix protein which binds to the
promoter of the insulin gene.
Bound DNA moves a lot slowly because mass has changed and charge has changed
Complex is dictated by the dissociation of the protein DNA interaction
- Only the edges of the base pairs
are accessible.
- The hydrogen bonding patterns
are different for the two grooves.
- In the minor groove the only
difference between GC and AT is the 2-
amino group of G
Methods for detecting interaction with DNA
1. Bandshifts- no good for weak interactions as the complex dissociates in the gel
- Bandshift experiments can be used to determine the sequence of the DNA- binding site
of a protein complex using a technique known as SELEX (systematic evolution of ligands
by exponential enrichment) This involves binding the protein of interest to a random
DNA sequence contained within a longer oligonucleotide template and separating the
bound and unbound DNA in a bandshift gel. The bound DNA is then amplified. The
consensus binding site is known after multiple amplifications.
- They can also be used to visualize protein- protein interactions between a DNA binding
protein and other non-DNA binding proteins in what is called a supershift assay in this
assay the binding of a second protein to a protein DNA complex is visualized by a
further retardation of mobility.
Examples is the use of antibodies to confirm the identity of a protein in a protein- DNA
complex as in the identification of a basic helix-loop-helix protein which binds to the
promoter of the insulin gene.
Bound DNA moves a lot slowly because mass has changed and charge has changed
Complex is dictated by the dissociation of the protein DNA interaction
