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
T/F: direct contact can be achieved even when promoter and regulatory protein’s binding sites are far apart
T; DNA looping
does euk RNAP recognizing binding site on its own?
no, needs general transcription factors
RNA pol I vs II vs III
RNAP I - rRNA
RNAP II - mRNA, snoRNA, snRNA, miRNA
RNAP III - tRNA, 5s rRNA, some snRNA
what 5 subunits are common to all 3 RNAPs
Rpb 5, 6, 8, 10, 12
RNAP II structure
core subunits: Rpb1,2,3 (similar to beta’, beta, and 2 alpha E.coli subunits)
functions of Rpb1, 2, 3
Rpb1 binds DNA
- 2 forms of large subunit: phosphorylation on carboxy-terminal domain (CTD)
– II alpha - non-phosphorylated form
– II o - phosphorylated form
– functionally diff: alpha binds to promoter, o is in elongation phase
Rpb2: polymerization active site
Rpb3: 20 AA region like bacteria subunit alpha
CTD tail
- not in RNAP I or III
- 7 repeated AAs (Tyr-Ser-Pro-Thr-Ser-Pro-Ser)
- hydrophilic, phosphorylation site
- critical for viability
- unphosphorylated CTD tail used to start transcription
- phosphorylated CTD present only for high lvls of transcription
- CTD tail critical for methyl cap addition and polyadenylation; splicing
nothing resembling operons is known in euk except?
nematodes
in studies, what is the problem with steady-state transcript levels
cannot distinguish between transcription rate and degradation rate of mRNA
reporter genes
- code for easily assayed gene products (usu enzymes)
- clone reporter gene after promoter of interest
- level of expression of reporter gene is proportional to strength of promoter
- can delete/mutate a part of promoter start from 5’ end (5’ deletion series) to map importance of seq in promoter
TATA-less promoters
in housekeeping genes or specialized genes (made only in certain cells)
- these genes NEED either initiator (core element) or GC boxes (upstream elements) to start transcription
____ from TATA to start is most important
distnace
functions of a TATA box
- TATA box locates start of transcription ~30 bp downstream
- sometimes important for transcription efficiency
- TATA-binding protein (TBP) binds to TATA box and starts assembly of general transcription factors and RNA pol
core promoter parts (3)
initiator element
- strength of promoter determined by surrounding nucleotide seq
- transcription with initiator elements or TATA boxes begins at precise site
downstream element
- binds TFIID (general TF)
TFIIB recognition element
- binds TFIIB (general TF)
- considered part of regulatory elements
regulatory elements (list)
GC boxes
- need to be close to TATA box, usu in housekeeping genes
- can be part of TATA-less promoter
– result: mRNAs with multiple alternative 5’ ends (UTRs)
CCAAT boxes
- enhancer upstream
- factor is CTF
- no prok equiv
promoter-proximal elemtns
- control regions upstream from start site
- enhance or repress
- cell-type specific
enhancers
- control elements that usu stimulate transcription
- activators bind here
enhancers are ___-independent
orientation (flipped 180 deg, same effect)
can enhancers occur anywhere?
yes, downstream in an intron or in exon
transvection in enhancers
enhancers on adjacent chromosomes (“local conc of factors”)
negative enhancer =?
silencer
class II promoters have 2 parts:
core elements + regulatory elements
what do core elements/ core promoter/ promoter include?
TATA box
initiator
downstream element
TF II B recognition element
