class 3 TFs and enhancers

two domains of TFs

dna binding domain

activation domain - controls level of trans. by recruiting pol II or cofactors

sequence specific TFs

interact with enhancers

bind specific DNA seqs.

also bind cofactors to faciliatate recruitment of more general TFs and Pol II

transcriptional cofactors

dont bind DNA directly

recruited by sequence specific TFs

mediator

important trans. cofactor

interact with seq sepcfic tfs and pol II

also helps sitmulate phos of ser5 (which helps pol II leave promoter complex)

cooperative TF binding

binding of one TF to an enhancer helps second one bind to the enhancer to upreg. trans.

can also be heterodimers that bind together first then together bind dna.

cobinding of TFs

when an intermediary (cofactor like mediator) facilitates binding of both TFs to DNA

co occupany tf binding

two tfs comepte for one enhancer binding site and they are constantly switching. gives rapid fine tuning of gene expression

pioneer tfs

can displace a nucleosome to open up an enhancer so that other tfs can bind to the enhancer

what does chip seq do and how does it work

you use it to find where in the genome is bound by proteins.

does this by first crosslinking proteins to the dna with formaldehyde

then cleave dna into pieces

then use antibodies to isolate proteins (like histones) or interest

then isolate those fragments that are bound to the histones

then heat it up to detach histones from dna.

then sequence those dnas

difference between rna seq and rt pcr

rna seq uses reverse trans. to find all of the active genes in a sample.

rt pcr only works on specific primers that you pre design and use those to search for them in a sample to detect just that sequence or gene.

enhancer reporter

method to identify and characterize DNA enhancer sequences

if reporter expression is observed then the enhancer is sufficient to drive transcription

get a plasmid with an enhancer candidate for a core promoter and reporter gene.