Lecture 9 - Proteins that regulate transcription Flashcards
2 methods that can detect protein - DNA interactions
EMSA and DNase I footprinting
What binds enhancers
TFs (transcriptional control elements) -> regulatory PROTEINS
4 techniques for identifying regulatory proteins that bind to DNA
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 **
What can EMSA/gel shift assays be used for ?
detect DNA binding proteins during biochemical purification
What can DNase I footprinting help for ? (2)
1) Reveal specific binding sites for DNA binding proteins
2) Assay for transcription factor purification (check if purification successful)
Co-transfection assays explanation
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.
Other method for knowing if a protein is a transcription factor for a particular gene
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)
EMSA is better than DNase I footprinting for ______________
quantitative analysis of DNA-binding proteins
What EMSA does not provide
Specific DNA-binding sequence
Logic of EMSA
Segment of DNA bound to a protein will migrate slower in a gel than DNA alone
what do we mean by bandshift or mobility SHIFT
Protein bound strand is retarded
Principles of liquid chromatography : different separations are based on what and usual order
Usually 1) Based on charge (Ion-exchange chrom.) 2) Based on size (gel filtration chrom.) 3) Based on affinity (Antibody-affinity chrom.)
Where is EMSA done (what technique) 3 things to note + why a certain technique is better
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)
EMSA done in vivo or in vitro
in vitro
Particularities of DNA used in EMSA (2)
1) Radiolabelled 2) Contains known regulatory element
EMSA control is sample of protein or DNA ? what do you incubate w/ what
Control is sample of the protein. In exp., protein is incubated with radiolabelled DNA (DNA probe)
DNase I footprinting important similarity with EMSA
Protein incubated with DNA probe
DNase I footprinting logic
If a protein is bound to a particular DNA sequence, it will protect it from nuclease digestion. (ex. DNase I endonuclease digestion)
What is a nuclease (how it works exactly)
Enzyme that cuts nucleic acids at phosphodiester bonds level
DNase I footprinting protocol summary
Protein bound to radiolabelled DNA and this DNA is then cleaved with nuclease that cuts randomly.
What can DNase I footprinting find
Precise binding site of protein to DNA
Which one would be logically done first ? EMSA or DNase I footprinting
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
Footprinting experiment can be done with __________ other than _________
proteins. DNase I
DNase I footprinting in vitro or in vivo
in vivo
DNase I footprinting on what is it done (which technique)
gel electrophoresis (after digestion)
What you compare on gel electrophoresis after footprinting
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.
First thing you can do with footprinting when you visualize your electrophoresis mapping
Identify the specific sequences bound by transcription factors.
What did DNase I footprinting allow to identify concerning GTFs and what did it use ?
Which sequences TBP and TFIID bind to in the proximity of a transcription start site.
How TFIID and TBP regions determined
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
Second thing you can do with footprinting when you visualize your electrophoresis mapping
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
Transcription factors what they are and what they bind to
Prots –> transcr. regulatory elements. Bind to promoter-proximal elements and enhancers.
Transcription factors particularity (special kind of protein)
Modular proteins. Contain a SINGLE DNA binding motif and one or more activation domains (for activators) or repression domains (for repressors)
Example of transfection assay for transcription activity in vitro
Transcription factor SP1 and promoter/enhancer of SV40 vs promoter enhancer of other virus like adenovirus.
What do we see on gel electrophoresis of SP1 that was added for adenovirus DNA transcription and SP1 that was added for SV40 transcription
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
Co-transfection assays (assay for transcription activity IN VIVO) explanation
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.
Co-transfection assays can identify __________ as well as __________
activators. repressors
Co-transfection assay : what condition for the cultured cell that is used ?
Must lack or not express the gene for the protein X that is tested
What does modular protein mean ?
Protein that has DISTINT FUNCTIONAL DOMAINS that can fold and be active independently of the rest of the protein
Transcriptional activators what they have (2 domains)
DNA binding domain and Activation domain that binds other proteins to stimulate transcription
What experiment was able to demonstrate modular characteristic of transcription activators
Exp. with lacZ gene and UASgal (and Tata box was the promoter)
What is UASgal
17 bp enhancer that binds transcr. activator called GAL4
What was done during exp. showing modular charact. of transcr. activator
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.
What was measured during exp. showing modular charact. of transcr. activator
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
what is B-galactosidase
Protein expressed by lacZ reporter gene
what happens if DNA-binding domain is cut (GAL4)
No binding to UAS. No ß-galactosidase activity
What happens if you delete parts of activation domain (GAL4)
Less ß-galactosidase activity
Internal deletion mutants of GAL 4 -> DNA binding domain and Activation domain are linked together
UASgal binding and ß-galactosidase activity are conserved except if you delete parts in the binding and activation domain
What can internal region in Gal4 (or internal regions in any modular proteins) serve for ?
Make prot flexible. Domains can move around
Which type of experiment proves modular nature of TFs
Domain-swapping experiments
What domain swapping experiments show
DNa-binding site from one transcr. activator can be matched with activation domain of another transc. activator and give functional protein
Transcriptional repressors freq./how much they occur
occur less than activators. much smaller pool of repressors
Difference with transcr. activators
have repressor domains
Repressors in prokaryotes -> what they do
Block Pol binding physically (sterically)
Repressors in eukaryotes -> what they do
Bring elements that will shut down DNA
What would be the control region of a gene
Region upstream of +1 start site containing promoter-proximal elements and all enhancer/repressor sequences
Concerning the control region of a gene, how does a particular cell type express or not express a gene
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.
