What are the proportions of different nucleotides at the mRNA start site?
- A - 50%
- G - 25%
- C,U - 25%
What is the TATA box? Where is it (give specific region)
TATA box is upstream of transcriptional start site in the -35 to -25 region
The TATA box indicates direction of transcription and DNA strand to be read
What are the 3 main elements characterized around the TATA and where are they?
Initiator - around start site
Bre - upstream of TATA box
DPE - downstream of promoter element and start site
What is a transcriptional control element?
Name 4
Promoter region is full of elements that confer specificity around the start site
- Exon
- Intron
- UAS (yeast enhancer)
- Promoter-proximal element (close to start site)
What is the UAS?
A yeast enhancer that is upstream or downstream of element that ensures specificity of transcription
- Can be up to 50 kb away
- Can even be on another chromosome (doesn’t have to be in cis)
What is a CpG?
Some genes use CpG as a promoter, it is in the promoter region and drives gene transcription in both directions (one is more stable than the other)
What is purpose of recombinant DNA technology?
Allows us to study transcription regions
How do we perform recombinant DNA technology?
How do we do it differently depending on the type of cell
Clone DNA into a plasmid to amplify the fragment, then introduce it into a host
- In bacteria, we perform transformation
- In mammalian cells, we perform transfection
- In animals/plants, we perform transgenics
What is transformation in recombinant DNA technology?
- Insert plasmid vector of DNA fragment to be cloned together (makes recomdinant plasmid)
- Place E.coli plates, then add ampicillin - DNA fragment will survive
- Perform cell multiplication, we mpw have multiple copies of DNA fragment
What is transfection in recombinant DNA technology?
Introduce plasmid vecotr into mammalian cells
- plasmid with specialized region will be ‘expression vector’
-
What is transfection in recombinant DNA technology?
Introduce plasmid vecotr into mammalian cells
- plasmid with specialized region will be ‘expression vector’
-
What methods can we use to understand the impact of upstream regions?
- 5’ deletion series
- Linker scanning analysis
How do 5’ deletion series work?
- Slowly cut down from 5’ end getting closer and closer to initiator
- introduce sections into a plasmid vector
- traqnsfect into cells with a reporter enxyme
- The reporter gene will allow us to quantify where important elements were dropped
What are 5 common reporter genes?
- GFP
- B galactosidase
- Thymidine kinase
- Luciferase
- Choarmophenicol acetyl transferase
Explain linker scanning analysis
- Use reporter as proxy foor transcription ability
Find restriction enzyme sites - take out different regions of upstream region to see which ones are important
When taking out a section and the reporter gene shows negative, this means we took out a section that has an important role
How does UAS ensure specificity from such a high distance away?
UAS directs RNA pol II, telling it where to go and at what time
- It gives tissue specificity
How can a Pax6 gene be used to determine importance of different regulatory elements?
Introduce Pax6 gene in different upstream areas, depending on how reporter gene is expressed we can see what is conserved.
This shows what our important regulatory elements are?
How can important regions be so far apart from each other?
Chromosomes form loops, resulting in regions becoming physically adjacent despite being linearly distant
The loops chomosomes form are often associated wtih active transport
