BIOC2306 transcription factors

How do DNA binding factors bind DNA

dont need access bases directly, dont need unzip

recognise atoms of major and/or minor groove that correspond to bases below it

diff aas H bond to bases in major and minor groove

which DNA groove contains more information

major groove

is recognit by a lot more binding proteins

what bases do Asn and Arg bind

A and G respectively

how many contacts are at TFprotein/DNA interface and what dose this determine

10-20

det. strength of binding and specificity

structure of helix-turn-helix DNA binding motif

C term DNA recognition DNA helix binds major groove

N term helix helps position recognition helix

proteins with HTH often bind as dimers

how does doubling the number of contacts of a HTH protein affect the affinity constant

squares the affinity constant

how many turns of the DNA helix separates the binding half sites of HTH protein dimers

one turn of helix

what are homeodomain proteins

TFs- essential regulatory proteins in eukaryotes

structure of HTH found in homeotic/homeodomain proteins

60 aa

helix 3 = DNA recognition helix

helix 1 and 2 = provide structural context

N term extension before helix 1 binds minor groove

two types of Zinc finger DNA binding domain

cys2-cys2

cys2-his2

structure of zinc finger domain

2 antiparallel beta strands with alpha helix

alpha helix binds major groove

zn coordinates 4 aas to hold one end of helix to end of a beta sheet

cys2-cys2 vs cys2-his2

his2 fingers occur in tandem, forming continuous stretch of alpha helixes contacting major groove, binding G, dont form dimers like cys2

cys2 dimers dimerise via c term alpha helix in each (mediates protein-protein interactions), N term contact DNA (det. specificity)

how far separated are the two halves of the recognition site in Zinc finger cys2-cys2 dimers

1 helix turn apart

do all of the zinc fingers of a TF have to bind DNA

no

what type of sequences do zinc fingers bind

palindrome (dyad symmetry)

halves separated by diff no of bases in middle

does zn in zinc finger contact the DNA

no, just coordinates 4 aas to hold end of alpha helix to end of one of beta strands

steroid receptors are are dimers of what

cys2-cys2 zinc fingers

what are 3 domains of steroid receptors

DNA binding

Hormone binding

transc reg domain

what type of experiments are used to determine function of a domain in a protein e.g. steroid receptor

domain swap experiments

which TFs must be dimers to bind DNA

leucine zippers

basic helix loop helix

structure of leucine zipper

L on one side of helix of each protein in dimer that form Hphobic interactions (zipper)

L every 7 aas in helix so on same side

each monomer has DNA binding domain

how does heterodimerisation within leucine zippers affect activity

can change affinity, binding specificity

can inactivate it

structure of basic helix loop helix

1 short helix inv in dimerisation

1 long helix inv in DNA binding

how can Helix-loop-helix proteins be inactivated by heterodimerisation

form dimer with protein that does have functional binding domain so cant bind

what does obligate dimers mean

protein never found as a monomer

what are zinc finger nucleases

a designer transcription factor

get many fingers in a row, each with 3 base specificity, so can design them to recognit any sequence

hook Fok1 endonuclease to it, cause cleavage of DNA where bind

downsides to zinc finger endonucleases

zn fingers that bind each triplet not all found in nature

some dont recognise triplet specifically

for Fok1 cutting, req protein to recognise palindromic sequence as a dimer

what is the TALE system

designer transcription factor

34 aa repeats, can be linked, where aa 12 and 13 specifiy the base that is recognised

so can design to recognise any seq

but now CRISPR-Cas9 system used to target specific DNA seqs

how do TFs work

stimulate activity of basal promoter complex

how do TFs synergise

diff TFs bind a same gene by diff mechanisms

e.g. enhancer recruitment, stim RNA pol, stim TFIID

what domains do all TFs have

DNA binding domain

transc activation domain

(obsv. in modules in gene encoding them)

what experiments can be used to id. functionality of domain of TFs

domain swap experiments

3 types of activation domains of TFs

acidic

glutamine rich

proline rich

how does activation domain of TFs increase transc

contact diff TFs/Trans acting factors in TFIID complex

via TFIIB (inc. rate of binding of TFIIB or its efficiency recruiting other components)

int. with mediator complex (cont. pol II CTD or TFIIH which cont and pi CTD, cause release of pol II)

can recruit co-activators (and co-repressors)

what is p300/CBP

co activator that many TFs interact with

directly contacts basal machinery or function to open chromatin (has HAT activity, open structure form)

if co-activators can recruit HATs then what can corepressors recruit

HDACs

do activators directly contact RNA pol II

No, do it indirectly via mediator complex (RNA pol CTD or TFIIH activation that pi CTD)