DNA binding proteins and euk transcription Flashcards

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1
Q

gel shift assays
(electrophoretic mobility shift assay (EMSA))
- principle

A

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

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2
Q

gel shift assays
(electrophoretic mobility shift assay (EMSA))

general steps

A
  • labeled olignucleotide probe, protein + probe, specific antibody + protein + probe
  • put in sample wells in denaturing polyacrylamide gel
  • autoradiography on x-ray film
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3
Q

DNase I footprinting theory

A
  • 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)
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4
Q

DNase I footprinting general steps

A

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
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5
Q

what do DNA-binding proteins “read” when looking for place to bind?

A

protein structures allow AAs to form regions that “read” DNA seq = DOMAINS

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6
Q

what do domains recognize?

A

domains recognize H bond acceptors (A; oxygen and nitrogen ions) and donors (D; hydrogen bond to acceptor)

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7
Q

maximum protein to DNA contact when DNA is _____

A

distorted;
these proteins can bind and bend DNA toward or away from bound dimer

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8
Q

domains definition

A

tertiary structure of large proteins organized in distinct regions; each domain is responsible for diff functions

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9
Q

motifs definition

A

specific combinations of secondary structures, which are organized into specific 3D structure inside domains that account for the function

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10
Q

transcription factor domains (3)

A

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

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11
Q

helix-turn-helix motif in DNA binding domain of prok TFs

A

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

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12
Q

binding sites for activators are called ____

A

enhancers

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13
Q

some proteins are regulators, meaning

A

can execute positive or negative regulation dep on presence/absence of effector
-> diff conformation = affinity for diff cis elements

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14
Q

control of initiation of transcription in E. coli is regulated through?

A

two component regulatory system

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15
Q

T/F DNA bending (due to binding of trans factor) can lead to negative OR positive regulation

A

T

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16
Q

T/F: direct contact can be achieved even when promoter and regulatory protein’s binding sites are far apart

A

T; DNA looping

17
Q

does euk RNAP recognizing binding site on its own?

A

no, needs general transcription factors

18
Q

RNA pol I vs II vs III

A

RNAP I - rRNA
RNAP II - mRNA, snoRNA, snRNA, miRNA
RNAP III - tRNA, 5s rRNA, some snRNA

19
Q

what 5 subunits are common to all 3 RNAPs

A

Rpb 5, 6, 8, 10, 12

20
Q

RNAP II structure

A

core subunits: Rpb1,2,3 (similar to beta’, beta, and 2 alpha E.coli subunits)

21
Q

functions of Rpb1, 2, 3

A

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

22
Q

CTD tail

A
  • 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
23
Q

nothing resembling operons is known in euk except?

A

nematodes

24
Q

in studies, what is the problem with steady-state transcript levels

A

cannot distinguish between transcription rate and degradation rate of mRNA

25
Q

reporter genes

A
  • 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
26
Q

TATA-less promoters

A

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

27
Q

____ from TATA to start is most important

A

distnace

28
Q

functions of a TATA box

A
  • 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
29
Q

core promoter parts (3)

A

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

30
Q

regulatory elements (list)

A

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

31
Q

enhancers are ___-independent

A

orientation (flipped 180 deg, same effect)

32
Q

can enhancers occur anywhere?

A

yes, downstream in an intron or in exon

33
Q

transvection in enhancers

A

enhancers on adjacent chromosomes (“local conc of factors”)

34
Q

negative enhancer =?

A

silencer

35
Q

class II promoters have 2 parts:

A

core elements + regulatory elements

36
Q

what do core elements/ core promoter/ promoter include?

A

TATA box
initiator
downstream element
TF II B recognition element

37
Q
A