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DNA-binding Domains Structural considerations of the DNA double helix Families of DNA-binding proteins. DNA-binding domains : Let’s look at DNA first. Heparin can be used to purify DNA-binding proteins. Virtually ALL DNA-binding proteins have intrinsic affinity to the phosphate
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DNA-binding Domains • Structural considerations of the DNA double helix • Families of DNA-binding proteins
Heparin can be used to purify DNA-binding proteins Virtually ALL DNA-binding proteins have intrinsic affinity to the phosphate backbone of DNA (i.e. they have a non-sequence specific affinity for DNA)
The minor groove harbors little chemical Information G:C C:G A:T T:A
The Helix-Turn-Helix motif was reinvented multiple times Homeodomain (yeast MatA1) TEA domain (human TEF-1) Anbanandam A et al. PNAS 2006;103:17225-17230
A (famous) mutation in a homeotic gene: Ultrabithorax Wild type Ultrabithorax From: Lawrence, The making of a fly
Basic Region Leucine zipper (bZIP) Dimer of two large a-helices that form a coiled coil Examples: FOS and JUN
Basic Region Helix-loop-Helix domain (bHLH) Example: MyoD
The C2H2 zinc finger domain Example: TFIIIA
Nuclear Receptors bind DNA via a pair of C4 zinc fingers Four cysteines are complexed with a Zn++ ion
Structural properties of zinc fingers binding to DNA C2H2 type C4 type (nuclear receptors)
Experimental tools to define regulatory DNA elements Testing function: Mutations/Promoter bashing Testing binding: DNAse I footprinting EMSA: Electrophoretic Mobility Shift Assay chIP: chromatin Immunoprecipitation
Defining promoter elements via promoter bashing ( ) * Expression Reporter Gene upstream region +++++ +++++ +++++ ++++ ++++ +++ + * + (+) - + +(+)
DNAse I footprinting • Idea: DNA-bound protein protect • DNA from digestion • Radiolabel DNA fragment at one end • Incubate protein with DNA • Digest DNA with low concentration • of DNAse I • Run fragments on high resolution gel • Map location of “footprint” based on • size of fragments
- Electrophoretic Mobility Shift Assay I + antibody + + + + labeled DNA + + + protein + “cold” competitor DNA +
Electrophoretic Mobility Shift Assay II Cold specific competitor DNA: usually a fragment of DNA, always unlabeled, such as a double-stranded oligonucleotide that contains the suspected DNA recognition sequence or mutations thereof. Typically, 10-100fold molar excess is used. If higher amounts are needed, the complex in question is likely to be non-specific. Non-specific competitor DNA: Because every DNA-binding protein exhibits non-specific affinity for DNA, one needs to add high amounts of non-specific DNA to saturate this activity. This is particularly important when using nuclear extracts. Most often, one uses synthetic DNA, such as poly [d(I-C)]. DNA-binding proteins: can be added in the form of crude nuclear extracts, biochemically purified proteins or in-vitro translated proteins.