Lecture 9 - Proteins that regulate transcription

Question 1

2 methods that can detect protein - DNA interactions

Answer 1

EMSA and DNase I footprinting

Question 2

What binds enhancers

Answer 2

TFs (transcriptional control elements) -> regulatory PROTEINS

Question 3

4 techniques for identifying regulatory proteins that bind to DNA

Answer 3

1) Biochemical purification by column chromatography
2) DNase I footprinting
3) Electrophoretic mobility shift assay (EMSA or gel shift)
4) Assay for effects on transcriptional activity **CAN BE DONE IN VIVO OR IN VITRO **

Question 4

What can EMSA/gel shift assays be used for ?

Answer 4

detect DNA binding proteins during biochemical purification

Question 5

What can DNase I footprinting help for ? (2)

Answer 5

1) Reveal specific binding sites for DNA binding proteins

2) Assay for transcription factor purification (check if purification successful)

Question 6

Co-transfection assays explanation

Answer 6

Co because you introduce in cultured cells a plasmid vector with Protein X gene AND reporter gene (that is to be transcribed). Useful to see if protein X is a transcription factor for reporter gene.

Question 7

Other method for knowing if a protein is a transcription factor for a particular gene

Answer 7

Incubate protein with DNA molecule containing gene + sequences upstream that contain enhancer. See if protein binds to DNA. (compare w/ a column that doesn’t have the protein)

Question 8

EMSA is better than DNase I footprinting for ______________

Answer 8

quantitative analysis of DNA-binding proteins

Question 9

What EMSA does not provide

Answer 9

Specific DNA-binding sequence

Question 10

Logic of EMSA

Answer 10

Segment of DNA bound to a protein will migrate slower in a gel than DNA alone

Question 11

what do we mean by bandshift or mobility SHIFT

Answer 11

Protein bound strand is retarded

Question 12

Principles of liquid chromatography : different separations are based on what and usual order

Answer 12

Usually 1) Based on charge (Ion-exchange chrom.) 2) Based on size (gel filtration chrom.) 3) Based on affinity (Antibody-affinity chrom.)

Question 13

Where is EMSA done (what technique) 3 things to note + why a certain technique is better

Answer 13

1) Example in notes = Column chromatography. Wiki = gel electrophoresis (polyacrylamide or agarose gel). 2) You can do it with any of both because shifting will happen because of prot binding w/ DNA and that’s the principle of EMSA 3) Column chromatography is more advantageous if you want to isolate the protein afterwards (ex. if you had a mixture of proteins and you want to isolate specifically the ones binding to the probe and calculate how much you had)

Question 14

EMSA done in vivo or in vitro

Answer 14

in vitro

Question 15

Particularities of DNA used in EMSA (2)

Answer 15

1) Radiolabelled 2) Contains known regulatory element

Question 16

EMSA control is sample of protein or DNA ? what do you incubate w/ what

Answer 16

Control is sample of the protein. In exp., protein is incubated with radiolabelled DNA (DNA probe)

Question 17

DNase I footprinting important similarity with EMSA

Answer 17

Protein incubated with DNA probe

Question 18

DNase I footprinting logic

Answer 18

If a protein is bound to a particular DNA sequence, it will protect it from nuclease digestion. (ex. DNase I endonuclease digestion)

Question 19

What is a nuclease (how it works exactly)

Answer 19

Enzyme that cuts nucleic acids at phosphodiester bonds level

Question 20

DNase I footprinting protocol summary

Answer 20

Protein bound to radiolabelled DNA and this DNA is then cleaved with nuclease that cuts randomly.

Question 21

What can DNase I footprinting find

Answer 21

Precise binding site of protein to DNA

Question 22

Which one would be logically done first ? EMSA or DNase I footprinting

Answer 22

EMSA first and then footprinting. Now that you determined which DNA sequence the protein binds, or vice versa, you want to determine where exactly it binds it

Question 23

Footprinting experiment can be done with __________ other than _________

Answer 23

proteins. DNase I

Question 24

DNase I footprinting in vitro or in vivo

Answer 24

in vivo

Question 25

DNase I footprinting on what is it done (which technique)

Answer 25

gel electrophoresis (after digestion)

Question 26

What you compare on gel electrophoresis after footprinting

Answer 26

You see nucleotides hybridized to labelled probes so you compare A) nts that you got hybridized with nts from a sample of labelled DNA that had no protein with B) nts that you got hybridized with nts from a sample that did have protein.

Question 27

First thing you can do with footprinting when you visualize your electrophoresis mapping

Answer 27

Identify the specific sequences bound by transcription factors.

Question 28

What did DNase I footprinting allow to identify concerning GTFs and what did it use ?

Answer 28

Which sequences TBP and TFIID bind to in the proximity of a transcription start site.

Question 29

How TFIID and TBP regions determined

Answer 29

On gel electrophoresis, Different mixes (word fraction is used …) containing A) Labelled DNA from sequence -50 to +50 in a particular gene B) different quantities of TBP and TFIID or no TBP/TFIID at all, incubated with DNase I. On electr. mapping, you see nucleotides that weren’t hybridized (are missing) from mixes w/ TBP/TFIID by comparing to control

Question 30

Second thing you can do with footprinting when you visualize your electrophoresis mapping

Answer 30

Identify fractions containing the transcription factors (after a column chromatography containing DNase I, labelled DNA and the TF) - Note : At the same time you can (maybe) identify the exact nts (sequence) TF was bound to by comparing w/ fragments in fractions that didn’t have the TF

Question 31

Transcription factors what they are and what they bind to

Answer 31

Prots –> transcr. regulatory elements. Bind to promoter-proximal elements and enhancers.

Question 32

Transcription factors particularity (special kind of protein)

Answer 32

Modular proteins. Contain a SINGLE DNA binding motif and one or more activation domains (for activators) or repression domains (for repressors)

Question 33

Example of transfection assay for transcription activity in vitro

Answer 33

Transcription factor SP1 and promoter/enhancer of SV40 vs promoter enhancer of other virus like adenovirus.

Question 34

What do we see on gel electrophoresis of SP1 that was added for adenovirus DNA transcription and SP1 that was added for SV40 transcription

Answer 34

More mRNA hybridized to probes of the gene when SP1 added for SV40 DNA transcription but no difference when it’s added for Adenovirus DNA transcription. SP1 specific to a particular sequence (promoter/enhancer) upstream of gene in SV40 virus

Question 35

Co-transfection assays (assay for transcription activity IN VIVO) explanation

Answer 35

Plasmid 1 (gene coding Protein X / TF) + Plasmid 2 (reporter gene + enhancer/reg. sequence) put together in a cultured cell. See if transcription of reporter gene happens.

Question 36

Co-transfection assays can identify __________ as well as __________

Answer 36

activators. repressors

Question 37

Co-transfection assay : what condition for the cultured cell that is used ?

Answer 37

Must lack or not express the gene for the protein X that is tested

Question 38

What does modular protein mean ?

Answer 38

Protein that has DISTINT FUNCTIONAL DOMAINS that can fold and be active independently of the rest of the protein

Question 39

Transcriptional activators what they have (2 domains)

Answer 39

DNA binding domain and Activation domain that binds other proteins to stimulate transcription

Question 40

What experiment was able to demonstrate modular characteristic of transcription activators

Answer 40

Exp. with lacZ gene and UASgal (and Tata box was the promoter)

Question 41

What is UASgal

Answer 41

17 bp enhancer that binds transcr. activator called GAL4

Question 42

What was done during exp. showing modular charact. of transcr. activator

Answer 42

Different deletion series of the gene of the protein GAL4 where introduced in a first plasmid in co-transfection assays. 2nd plasmid was always UASgal and lacZ gene.

Question 43

What was measured during exp. showing modular charact. of transcr. activator

Answer 43

Binding of GAL4 to UASgal and ß-galactosidase activity (protein encoded by lacZ –> activity = activaiton domain is able to stimulate transcr. and works) in different cultured cells where co-transfection assays where done

Question 44

what is B-galactosidase

Answer 44

Protein expressed by lacZ reporter gene

Question 45

what happens if DNA-binding domain is cut (GAL4)

Answer 45

No binding to UAS. No ß-galactosidase activity

Question 46

What happens if you delete parts of activation domain (GAL4)

Answer 46

Less ß-galactosidase activity

Question 47

Internal deletion mutants of GAL 4 -> DNA binding domain and Activation domain are linked together

Answer 47

UASgal binding and ß-galactosidase activity are conserved except if you delete parts in the binding and activation domain

Question 48

What can internal region in Gal4 (or internal regions in any modular proteins) serve for ?

Answer 48

Make prot flexible. Domains can move around

Question 49

Which type of experiment proves modular nature of TFs

Answer 49

Domain-swapping experiments

Question 50

What domain swapping experiments show

Answer 50

DNa-binding site from one transcr. activator can be matched with activation domain of another transc. activator and give functional protein

Question 51

Transcriptional repressors freq./how much they occur

Answer 51

occur less than activators. much smaller pool of repressors

Question 52

Difference with transcr. activators

Answer 52

have repressor domains

Question 53

Repressors in prokaryotes -> what they do

Answer 53

Block Pol binding physically (sterically)

Question 54

Repressors in eukaryotes -> what they do

Answer 54

Bring elements that will shut down DNA

Question 55

What would be the control region of a gene

Answer 55

Region upstream of +1 start site containing promoter-proximal elements and all enhancer/repressor sequences

Question 56

Concerning the control region of a gene, how does a particular cell type express or not express a gene

Answer 56

it expresses the proteins that binds to the regions of interest in the control region. You want transcription = you express activators and they will bind to enhancers. You don’t want transcr … etc.