Lecture 8 - Regulatory sequences in protein coding genes Flashcards
What regulates expression of eukaryotic protein-coding genes ?
Multiple protein binding transcriptional control regions. (promoter-proximal elements, enhancers)
3 things that promoters do
1) Direct binding of RNAP II to DNA 2) Position RNAP II on transcription start site 3) Regulate frequency of transcription initiation (depending on how precise promoter is)
2 things that promoter-proximal elements and enhancers influence concerning gene expression
Influence 1) probability of gene expression 2) rate of gene expression
Promoter-proximal elements and enhancers are _________ specific
cell-type
Exemple of gene w/ multiple transcription-control regions
Pax6 gene in mouse
In a certain cell type, how can you express different forms of a gene that has multiple transcription-control regions
you express correct activators that will bind to a particular transcriptional-control region
what figure shows that we can control gene expression in a specific cell-type w/ activators that are specific to different transcriptional-control regions
Figure with 2 mice embryos. In one you see exon 1 (green) of pax6 gene -> so gene expressed in pancreas. In the other, you see orange exon between exons 4 and 5 –> rene expressed in retina
what does figure with 2 mouse embryos show
Diff. Transcriptional-control regions/promoters in Beta gal gene (reporter gene in this exp.) ALLOW CELL TYPE DIFFERENTIATION 1st image: If we insert green promoter/trans.control.reg., with the gene, we see gene expressed in pancreas 2nd image. If we insert orange promoter/trans.control.reg. w/ the gene, we see gene expressed in develop. retina. However, gene expressed in all cell types that have specific activators for it ….
pax 6 gene promoters or transcript. control regions . prof ?
Promoters (acc. to prof), says 200-500 bp …, activators. transcript. factors …
Promoter : term signification in this course
Tata box or other sequences that recruit RNAP to transcription start site
What is recombinant DNA ? and what is something particular you can do w/ that ?
DNA molecules formed by laboratory methods). Can bring together genetic material from multiple sources
What is a plasmid ? 3 things
1) DNA molecule distinct from chromosomal DNA and that can autoreplicate 2) Usually circular form 3) Contains at least one sequence which serves as replication origin.
What is a restriction enzyme ?
enzyme that cleaves at specific sequences in DNA
What is a vector ?
DNA molecule used as a vehicle to carry foreign genetic material into a cell where it can be replicated or expressed. Vector containing foreign DNA is termed as recombinant DNA
What are recombinant DNA libraries?/what does it mean/what do they represent ?
Represent all genes or mRNAs expressed in a CELL TYPE
What can recombinant DNA libraries be used for ?
Identify genes and Investigate gene product function.
DNA Cloning and characterization different steps
Vector + DNA fragment —> Recombinant DNA —> Replication of recombinant DNA within host cell —> Isolation, sequencing and manipulation of purified DNA fragment.
What is a marker (molecular marker) ?
molecule that is used to identify a particular sequence of DNA in a pool of unknown DNA.
Possible use of a marker (molecular marker) ?
After cleaving the gene of interest, introduce in the plasmid vector w/ a marker in order to identify easily the gene of interest within the vector
Exemple of restriction enzyme that cleaves DNA and particular sequences left after cleavage
EcoRI. On both strands, from 3’ to 5’, you have CTTAAG which bond (complementary) and enzymes cleaves between A and G on both strands. Left w/ 2 DNA helices that have C and G on the end + TTAA (sticky ends) attached to the C
How do you insert DNA fragment in plasmid vector?
Using enzymes
E. coli replication and multiplication w/ recombinant DNA step 1
1) Mix E.coli with plasmids in presence of CaCl2
E. coli replication and multiplication w/ recombinant DNA step 2
2) Heat-pulse culture on nutrient agar plates containing ampicillin
E. coli replication and multiplication w/ recombinant DNA step 3
3) Cells that do not take up plasmid die on ampicillin plate and Transformed E.coli cells (contain E.coli chromosome) survive
E. coli replication and multiplication w/ recombinant DNA step 4
4) Plasmid replication
E. coli replication and multiplication w/ recombinant DNA step 5
5) Cell multiplication : colony of cells, each containing copies of the same recombinant plasmid is obtained
what does DNA cloning in a plasmid vector permit
Amplification of a DNA fragment
Promoter-proximal elements what they are
Sequences within 100-200 bp of promoter that can regulate transcription
Enhancers what they are and their frequency in prokaryotes
Long distance (thousands of kb away from transcription start site) transcriptional control elements. Rare/few in prokaryotes
What is a linker-scanning mutation ?
Replacement of nts in potentially important regions by replacement w/ synthetic oligonucleotide of known sequence. (scrambled sequence)
What can linker-scanning mutations be used for ? Protocol ?
Identify promoter-proximal elements. Linker-scan overlapping sequences in control region of a gene in vector DNA and see level of gene expression.
Promoter-proximal elements are _______ and do not _______. Linker-scanning allows us to make difference between elements that are ________ for transcription and those who _______.
distinct. do not overlap. necessary. contribute.
Limitation of linker-scanning mutations for finding promoter-proximal elements
Experimental approach that is good for detailed analysis of a short stretch of DNA.
Where can enhancers be and usual distance. What binds to them
Can span many kilobases. Can be upstream of coding region. Downstream of the whole gene. Within gene (in an intron). Can also be reversed (flipped nts). Bound by transcription factors
What technique can identify enhancers
Deletion analysis
First step of deletion analysis.
Using recombinant DNA techniques and different cleaving enzymes, obtain a serie of segments upstream of the start site that have different lengths. NOTE : Not put in vector yet ! we only took them from the (original) DNA.
Particularity of DNA transcription reading in a vector.
Can be read from 5’ to 3’ (or 3’ to 5’)
If different series of segments obtained at 1st step of deletion analysis are 5’ of the start site, what do we call them
5’-deletion series
Second step of deletion analysis
Ligate 5’-deletion series into vector carrying reporter gene
Third step of deletion analysis
Transform E.coli (REPLICATE) and isolate plasmid DNAs
Fourth step of deletion analysis
Transfect each type of plasmid separetely into cultured cells
What is not to forget if we want to visualize effect of deletion analysis
We need control cell with the reporter plasmid (containing whole sequence upstream of gene/not deleted). It will express the reporter mRNA and the reporter enzyme.
Fifth step of deletion analysis
Prepare cell extract and assay ACTIVITY of reporter enzyme
How do we choose reporter gene for deletion analysis
We choose gene that encodes an easily measured enzyme (ex : beta-gal)
If we see difference in reporter gene expression between plasmid w/ x kb serie and plasmid w/ y kb serie, what does that mean ?
Enhancer between X and Y kb
What is not to forget in deletion analysis (analysis of enhancers) ?
Promoter-proximal elements are there and bound by proteins too
Limitation of deletion analysis
We can only identify elements that are CLONABLE in our vector of choice. Limit to the length of deletion series. Ex: bacteria won’t replicate a 100 kb serie in a plasmid vector
Can enhancers and promoter-proximal elements substitute each other ?
No. Enhancers can’t substitute promoter-proximal el. and vice versa. Enhancer can’t substitute another enhancer and same between diff. promoter-prox. el.
Enhancers distance (numeric) and what they are specific to
Can be >50 kb away from gene they regulate. Are often cell-type specific.
what is UAS
Upstream activating sequence : regulatory element in most yeast genes that acts like an enhancer
Tata box distance in yeast
90 bp upstream from the start site
One of first enhancers to be studied and how
SV40. Transfection assay. Plasmid 1 with ß-globin gene and SV40. Plasmid 2 : ß-globin gene only. More ß-globin mRNA is synthesized in cells containing plasmid 1.
Protocol for transfection assay illustrating activity of SV40 enhancer. Step 1
Transfect plasmid 1 or 2 into cultered fibroblasts
Protocol for transfection assay illustrating activity of SV40 enhancer. Step 2
Isolate RNA and hybridize with labeled ß-globin DNA probe
Protocol for transfection assay illustrating activity of SV40 enhancer. Step 3
Treat with S1 nuclease (endonuclease that degrades ssDNA or ssRNA)
Protocol for transfection assay illustrating activity of SV40 enhancer. Step 4
Perform gel electrophoresis and autoradiography
