Methods

Question 1

Describe DNA footprinting

Answer 1

one control fragment with the top strand labelled will have no protein bound to it and will be nicked at the proper location (where the restriction enzyme cleaves); the other dsDNA (top strand labelled) will have a protein bound to it at it’s specific sequence, and will prevent the restriction enzyme from cleaving there; the DNA will be denatured and visualized on a polyacrylamide gel; the control lane will be a “ladder” of fragment; and then you can compare you protein-bound DNA–>where there is no bands (or much less intense) will be where the protein has bound, so those spots could not be cleaved; then you will be missing strand of that certain length–you can then figure outw here the protein binds, and possibly the sequence if you’ve already sequenced the gene??

Question 2

what are the main interactions between a protein and DNA in groove binding?

Answer 2

vdW forces and H-bonds

Question 3

what are the major groups of DNA binding domains?

Answer 3

Helix turn helix, helix loop helix, zinc fingers, leucine zipper???

Question 4

HtH general info

Answer 4

two alpha helices–one fits into hte major groove for specific binding, the other is for stability–to the phosphate backbone

Question 5

what do homeoboxes bind?

Answer 5

HtH and act and transcription factors

Question 6

HtH protein binds to Hox gene?? causes expression of gene??–acts as transcription factor

Answer 6

ok

Question 7

zinc finger proteins general info

Answer 7

20-30 aas in length; most common zinc finger is the C2H2–>zinc is held by 2 cysteines and 2 histidines; specific binding along the major group but not just dicated by aa seq alone

Question 8

xenopus zinc finger

Answer 8

has 9 zinc fingers; fingers 1,2,3 and 7,8,9 bind the major groove–each triple finger binds a 14 bp seq–doesn’t need to be exact, just close enoguh

Question 9

leucine zipper general info

Answer 9

can only work as a dimer–dimer of alpha helices–may be a homo or heterodimer; n-terminus enriched for basic amino acids and will contact DNA; C terminus has repeating leucines, every 7th aa; binds to the front and back of major groove; bind about a 4bp seq

Question 10

how are the coiled coils stabilized in leucine zippers?

Answer 10

the hydrophobis leucines pair to each other and stabilize the structure

Question 11

HLH general info

Answer 11

“discontinuous leucine zipper”–>two alpha helices held together by amphipathic forces; quite often regulate genes as heterodimers