Lecture 16 (RR3): Gene Regulatory sequences in RNA Poll II promoters. Flashcards
TATA sequence
- TATA sequence directs transcription at the promoters of protein coding genes.
- Genes share some common nucleotide sequences in and around their transcriptional start sites. At position about -25 or -35 upstream of the transcriptional start site, there is a collection of nucleotides that seem to be conserved and were present in almost all industrial gene sequences.This sequence became recognized as the TATA box or TATA element. It is a DNA sequence that is important for the initiation of transcription.
What are the proximal “cis-acting’ elements driving transcriptional initiation?
Downstream initiator element: located right around the transcriptional start site (-2 to +4).
BRE or TFIIB recognition element: Slightly upstream of the TATA box.
Downstream promoter element: actually within the gene downstream of transcriptional start site. Contribute to transcriptional efficiency.
These CIS elements work approximately around the transcriptional start site, presumably to somehow direct the RNA polymerase machinery to the appropriate genes (so that they start at the right place and begging transcribing the gene correctly).
What transciptional control elements regulate eukaryotic genes?
Proximal Promoter elements will be around from the transcription start site up to 1000 base pairs upstream. Show here at around -200. These are referred to as proximal because they are relatively close to the sites that they will act upon.
Enhancers do not work proximally at all, they work very far upstream or downstream of their targets. Sometimes they dont even work in CIS, they can be on another chromosome and yet still specify transcription og a given target. They confer tissue specificity (MAIN ROLE) and temporal specificity (sometimes)
- Enhancers can be located thousands of base pairs away from a gene’s promoter, or even be located within a gene.
- Once bound to enhancers, the transcription factors can get in close proximity to the promoter.
- They recruit RNA polymerase, facilitating transcription.
**CpG Islands: **Very important in a lot of mammalian promoters. CpG rich promoters send polymerase in divergent directions, the polymerase will take off in one direction or the other. Why would the cell waste energy to send polymerase in the wrong direction?? The transcripts that are made in the right direction are stabilized and the ones made in the wrong direction are not.
- majority of mamalian genes do not have TATA box and instead have CpG islands
- Most yeast have distal sequences (like enhancers) called yeast UAS. These work further away from the TATA box (less proximal than mammalian enhancers).
What is the most efficient way to identify the DNA elements involved in affecting the transcriptional efficiency of a given gene?
**Recombinant DNA technology. **
- DNA cloning in a plasmid vector permits amplification of a DNA fragment. This can be introduced into a host for study. - - - Introduce DNA elements into a specific plasmid that has been designed so that you can find the appropriate expression (often need to grow lots of the plasmids first → introduce DNA bacteria to transform them).
They have different names for the process of introducing a vector into a cell depending on which type of cell it is:
Bacteria -> Transformation (image to the right)
Mammalian cells -> Transfection
Live animals/plants -> Transgenics
What is an expression vector?
Transient transfection is the process of introducing a vector into a mamalian cell.
Expression vectors: specialized vectors used to express a gene in an eukaryotic cell.
Then you can examine those genes that you are interested in in the specific cellular context.
How do you identify transcriptional regulatory regions?
GENE CLONING - PCR
*To identify where the elements are in the region of DNA, you use PCR. You PCR a region that is upstream of the gene you are interested in.
1) You make a PCR product that corresponds to a region downstream of the start site or upstream in the regulatory region.
2) Then you introduce that fragment into an expression vector to evaluate how well this regulatory sequence activates some gene downstream.
3) Each PCR derived amplicon corresponds to a smaller region of the larger regulatory region.
4) You introduce all these variations that correspond to smaller chunks of that regulatory region of the gene and you put them all upstream of the same type of reporter.
5) Then you can grow them up in bacteria to get lots and lots of DNA and introduce those expression vectors individually into the mammalian cells. One variant gets transfected into one group of cells, another variant gets transfected into another group of cells.
6) Then you assess the reporter gene activity, and the only differences that you should see are dependent on the regulatory regions or lack thereof. They were driving the expression of the reporter genes.
What is the best reporter gene? what is the role of the reporter gene? Name some common reporter genes.
- The reporter gene is introduced into a cell and can be used to measure the promoter activity. Reporter gene = relative quantification of transcriptional efficacy.
- The best reporter gene is GFP
- another reporter gene called luciferase comes from a firefly and gives off light. We can quantify the ammount of light that is given off and use it as a proxy for how well the gene was transcribed (other options: antibodies, colorimetric assays).
Some common “Reporters”
-GFP
-β-galactosidase (lacZ)
-thymidine kinase (tk)
-luciferase (luc)
-chloramphenicol
acetyltransferase (CAT)
Linkr Scanning Mutations
- Linker scanning mutations can be used to find cis-acting regulatory sites.
- In linker scanning mutagenesis, a region of eukaryotic DNA (tan) that supports high-level expression of a reporter gene (light purple) is cloned in a plasmid vector as diagrammed at the top.
- Overlapping linker scanning (LS) mutations (cross hatched areas = areas removed) are introduced from one end of the region being analyzed to the other. These mutations are created by scrambling the nucleotide sequence in a short stretch of the DNA. After the mutant plasmids are transfected separately into cultured cells, the activity of the reporter-gene product is assayed.
- In the example shown here, the sequence from −120 to +1 of the herpes simplex virus thymidine kinase gene, LS mutations 1, 4, 6, 7, and 9 have little or no effect on expression of the reporter gene, indicating that the regions altered in these mutants contain no control elements.
- Reporter-gene expression is significantly reduced in mutants 2, 3, 5, and 8, indicating that control elements (brown) lie in the intervals shown at the bottom. (b) Analysis of these LS mutations identified a TATA box and two promoter-proximal elements (PE-1 and PE-2).
Transcriptional control elements
Elements that are situated around the transcriptional start site that help guide RNA Polymerase where it should sit down and begin.
1) TATA Box: proximal element
2) CpG island containing promoters: most mammalian cells are much more complex and therefore have CpG islands instead of TATA boxes. These are the promoters in mammalian cells
3) Enhancers: DNA regulatory elements that act at a distance. Can we upstream or downstream. Come together in loops and can interact with key proteins to tell the polymerase where to sit down. Confer instructions.
- In yeast, they are referred to as upstream activating sequences (UAS), they are in fact upstream more.
Divergent transcription
divergent transcription = transcription in both directions (might be why 80% of the genome is transcribed). We don’t know why this happens but it happens often (it is more the rule than the exception).
Mammalian genes with TATA box vs CpG island promoter vs yeast
Mammalian genes with a TATA box promoter
- Regulated by promoter-proximal elements and enhancers.
- The promoter elements shown in Figure 8-11 position RNA polymerase II to initiate transcription at the start site and influence the rate of transcription.
- Enhancers may be either upstream or downstream and as far away as hundreds of kilobases from the transcription start site. In some cases, enhancers lie within introns.
- Promoter-proximal elements are found upstream and downstream of transcription start sites at equal frequency in mammalian genes.
Mammalian genes with a CpG island promoter: - Transcription initiates at several sites in both the sense and antisense directions from the ends of the CpG-rich region.
- Transcripts in the sense direction are elongated and are processed into mRNAs by RNA splicing. These genes express mRNAs with alternative exons determined by the transcription start site.
- Genes with CpG island promoters contain promoter-proximal control elements. Currently, it is not clear whether they are also regulated by distant enhancers.
S. cerevisiae genes
- contain only one regulatory region, called an upstream activating sequence (UAS), and a TATA box, which is about 90 bp upstream from the transcription start site.
Regulation of transcriptional activation relies on…
- Regulation of transcriptional activation relies on control regions that can be far away
Mouse PAX6 gene vs Human PAX6 gene
Mouse PAX6 gene → highly conserved gene.
* Plays a role in specifying the various regions of the brain (also retina, lens,pancreas).
* All the coloured regions and squares correspond to enhancer regions → they are driving the expression of that gene in those tissues.
* The enhancers are DNA elements that are critical in gene expression because they confer where a given gene is going to be expressed and very often, at what time during development it will be expressed.
Compare to Human PAX6 gene → great deal of similarity.
* There are 3 different transcripts, driven by 3 different promoters.
* The grey ovals are the highly conserved sequences among vertebrates, suggesting that they must play some important role.
* Colored ovals (highly conserved) can be copied and put into expression vectors. Use these expression vectors to make transgenic zebrafish (best vertebrate model). They express the reporter genes in the appropriate location that one would expect in the PAX gene expression in a completely different organism (zebrafish). **This means that the conserved sequences (the nucleotide sequences that make up these elements) are all functional across all of these different species. **
Enhancers
- Enhancers are cis-acting elements that control tissue-specific or stage-specific transcription.
- If you compare nucleotide sequences between organisms, you’ll see an extraordinary amount of variation. Nucleotide sequence is constantly being sensitized to changes, its fluctuating all the time.
- This shows that the nucleotide sequence has been altered from its initial sequence that might have been present in an early ancestor. But some still remain almost identical.
- If they are unchanged/conserved between organisms, it means tha they play a functional role (needed for an organisms survival).
- In the red box (SALL 1 gene) particular sequence in this gene that is HIGHLY conserved between organisms. Therefore, it must play a critical role in the function of SALL 1 gene.
- This gene is expressed exquisitely in the growing limbs. SALL 1 plays a role in growing limbs.
How do enhancers work when they work at a distance
- Genes are very flexible
- Chromosomes can form loops that result in regions that are linearly very distant to become physically adjacent to one another.
- When chromatin loops up, regions that seem to be linearly very distal end up being close in 3D space. So, Chromatin loops out when transcription takes place and are maintained by proteins.
- These loops are often associated with active transcription. Enhancers may help to generate, stabilize, and increase the rate of transcription within these loops, even if they are linearly very far from their targets.
