RR3 Flashcards
What is the function of the TATA box and where is it located?
The TATA box indicated the direction of transcription and DNA strand to be read. The TATA is upstream of the transcriptional start site, it has similar sequences near the start site and position (-35 to -25 position)
A TATA box is a DNA sequence that indicates where a genetic sequence can be read and decoded.
What are other sequences/elements that were characterized around TATA?
1) Initiator (around start site)
2) Bre - Upstream of TATA box
3) DPE - downstream promoter element
What are some transcriptional control elements?
1) Promoter regions are full of elements that confer specificity around start site
2) Exon (region that ends up inside mRNA)
3) Intron (spliced out because it is a non-coding region)
4) UASC (yeast enhancer) -> DNA element that ensures specificity
5) Promoter-proximal element (close to start site -200)
What is Enhancer UAS?
It is upstream of downstream element that ensures specificity of transcription. Can be up to 50 kb away, can be on other chromosome.
Name a promoter that can activate transcription of downstream genes:
CpG island - it is in the promoter region and drives gene transcription in both directions, one direction is more stable then the other.
What is the function of Recombinant DNA technology? What are the three types (for what)?
It is used to study transcription regions. You clone DNA into a plasmid to amplify the fragment and then introduce a host.
Bacterial cells - transformation
Mammalian cells - transfection
Live animal/plants - transgenics
How does transformation work? (Recombinant DNA technology)
It is used to study the transcription regions in bacterial cells.
1) Insert plasmid vector of DNA fragment to be cloned together which makes recombinant plasmid add ampr
2) Place E. coli plates (DNA fragment will survive) and then add ampialin.
3) Perform cell multiplication, now have multiple copies of DNA fragment.
How does transfection work? (Recombinant DNA technology)
This is used to study transcription regions in mammalian cells.
Introduce vector into mammalian cells. A plasmid with a specialized region will be ‘expression vector’
How do we discover the impact of a upstream region?
Make a 5’ deletion series of upstream regions to then identify the important control elements.
1) make the 5’ deletion series
2) Introduce into specialized plasmid (express reporter gene)
3) Transfect into cells individually
4) reporter enzyme x 5
Can quantify expression of each one (reporter-gene expression) and can now identify important control elements.
What is a isoform?
Proteins that are similar/have similar functions.
What is the function of reporter genes?
Relative quantification of transcriptional efficiency
How does linker scanning regions work? And what is it useful for?
It is useful when looking at bigger regions. Use reporter as a proxy (imprints created during past climate) for transcription ability. Find restriction enzyme sites that take out different regions to see which ones are important by looking at reporter gene expression.
What are some common reporter genes?
GFP
B galactosidase
Thymidine Kinase
Luciferase
Charamonophemid acetyl transferase
What is RNA Pol II?
Multiprotein complex that transcribes DNA into precursors of mRNA
How do enhancers elements actually direct RNA Pol II?
UAS gives tissue specificity.
