DNA binding proteins and euk transcription Flashcards
gel shift assays
(electrophoretic mobility shift assay (EMSA))
- principle
principle: electrophoretic mobility of naked DNA fragment and DNA fragment w/ proteins differ
- mix restriction digest
- look for “shifted” bands
- no denaturing bc need proteins to retain 3D structure to bidn to DNA
gel shift assays
(electrophoretic mobility shift assay (EMSA))
general steps
- labeled olignucleotide probe, protein + probe, specific antibody + protein + probe
- put in sample wells in denaturing polyacrylamide gel
- autoradiography on x-ray film
DNase I footprinting theory
- nuclease treatment under limiting conditions (e.g., low temp/enzyme) -> on avg, each copy of DNA fragment only cut once
- DNA (no protein) - ladder of fragment on gel, each labeled at end, at least one DNase cleavage site
- DNA (with protein) - blank area corresponds to protein footprint (phosphodiester bonds protected, DNase cant cut)
DNase I footprinting general steps
end-labeled DNA fragment in 2 samples:
1) no protein added
2) protein added
- both partial digestion by DNase I
- x-ray film shows where protein binds
what do DNA-binding proteins “read” when looking for place to bind?
protein structures allow AAs to form regions that “read” DNA seq = DOMAINS
what do domains recognize?
domains recognize H bond acceptors (A; oxygen and nitrogen ions) and donors (D; hydrogen bond to acceptor)
maximum protein to DNA contact when DNA is _____
distorted;
these proteins can bind and bend DNA toward or away from bound dimer
domains definition
tertiary structure of large proteins organized in distinct regions; each domain is responsible for diff functions
motifs definition
specific combinations of secondary structures, which are organized into specific 3D structure inside domains that account for the function
transcription factor domains (3)
DNA binding domain: responsible for binding DNA with structural motifs that read DNA seq
Effector binding domain: can be altered by binding of a small molecule or covalent modification at effector site; causes conformational change in TF
Oligomerization domain: allow specific dimerization w similar TFs
helix-turn-helix motif in DNA binding domain of prok TFs
most frequent motif in DNA binding domain of bacterial repressors = helix-turn-helix motif
- 2 short alpha helices connected with a short “turn” (recognition helix and stabilizing helix)
- DNA recognition helix makes most of the contact w DNA and the other stabilizes the interaction
binding sites for activators are called ____
enhancers
some proteins are regulators, meaning
can execute positive or negative regulation dep on presence/absence of effector
-> diff conformation = affinity for diff cis elements
control of initiation of transcription in E. coli is regulated through?
two component regulatory system
T/F DNA bending (due to binding of trans factor) can lead to negative OR positive regulation
T