Lecture 18 Flashcards
RNA pol I function
transcribes rRNA genes
18S, 28S, 5.8S
RNA pol II function
mRNAs transcribes proteins
RNA pol III function
makes tRNAs
(5S rRNA, small rRNAs)
TFIIIA
TFIIIA
binds to internal control region (ICR) and makes contact at specific sequences
Somatic 5S RNA genes
in the arms, legs, brain and skin
only 2% of 5S genes
Oocyte 5S RNA genes
during oogenesis
98% of 5S RNA genes (largely off in adults though)
DNAase footprints
footprints that DNA binding leaves on the DNA
How do you check for DNA footprints? (4 steps)
- Add a radioactive p32 label to DNA
- Add a DNA binding protein
- Add DNAase. DNAase will cut everywhere but where the binding protein is.
- Run on a gel to see where fragments are.
What are zinc fingers made of?
Zinc ions, 2 histidines, and 2 cystisines
How many zinc fingers does TFIIIA have?
9
Where do the zinc fingers insert?
The alpha helical part of the finger inserts into the major groove of the DNA
Site Directed Mutagenesis
target specific amino acids to determine their function.
Important to have subtle changes.
Change the experimenters made in the zinc fingers.
Histidine to Asparagine
His to Asn
How did they get the TFIIIA into the cell once mutated?
- Subcloned into cDNA sp6 vector
- Added NTPs, a Guanine cap, and 5’ cap
- This gave capped synthetic TFIIIA mRNA
- It was injected into fertilized egg at first cleavage
How did they verify stability of the injected RNA?
Northern blot
