DNA binding proteins and euk transcription

Question 1

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

Answer 1

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

Question 2

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

general steps

Answer 2

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

Question 3

DNase I footprinting theory

Answer 3

• 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)

Question 4

DNase I footprinting general steps

Answer 4

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

Question 5

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

Answer 5

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

Question 6

what do domains recognize?

Answer 6

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

Question 7

maximum protein to DNA contact when DNA is _____

Answer 7

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

Question 8

domains definition

Answer 8

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

Question 9

motifs definition

Answer 9

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

Question 10

transcription factor domains (3)

Answer 10

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

Question 11

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

Answer 11

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

Question 12

binding sites for activators are called ____

Answer 12

enhancers

Question 13

some proteins are regulators, meaning

Answer 13

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

Question 14

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

Answer 14

two component regulatory system

Question 15

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

Answer 15

T

Question 16

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

Answer 16

T; DNA looping

Question 17

does euk RNAP recognizing binding site on its own?

Answer 17

no, needs general transcription factors

Question 18

RNA pol I vs II vs III

Answer 18

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

Question 19

what 5 subunits are common to all 3 RNAPs

Answer 19

Rpb 5, 6, 8, 10, 12

Question 20

RNAP II structure

Answer 20

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

Question 21

functions of Rpb1, 2, 3

Answer 21

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

Question 22

CTD tail

Answer 22

• 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

Question 23

nothing resembling operons is known in euk except?

Answer 23

nematodes

Question 24

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

Answer 24

cannot distinguish between transcription rate and degradation rate of mRNA

Question 25

reporter genes

Answer 25

• 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

Question 26

TATA-less promoters

Answer 26

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

Question 27

____ from TATA to start is most important

Answer 27

distnace

Question 28

functions of a TATA box

Answer 28

• 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

Question 29

core promoter parts (3)

Answer 29

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

Question 30

regulatory elements (list)

Answer 30

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

Question 31

enhancers are ___-independent

Answer 31

orientation (flipped 180 deg, same effect)

Question 32

can enhancers occur anywhere?

Answer 32

yes, downstream in an intron or in exon

Question 33

transvection in enhancers

Answer 33

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

Question 34

negative enhancer =?

Answer 34

silencer

Question 35

class II promoters have 2 parts:

Answer 35

core elements + regulatory elements

Question 36

what do core elements/ core promoter/ promoter include?

Answer 36

TATA box
initiator
downstream element
TF II B recognition element