HC3 regulation of gene expression

Question 1

prokaryotic transcription + RNA codes for?

Answer 1

• RNA codes for several proteins = polycistronic.
• no nucleus, so transcription and translation happen simultaneously.
• RNA pol binds directly to the promotor.
• Regulator proteins bind on/near RNA pol binding site

Question 2

eukaryotic transcription + modifcation of RNA

Answer 2

• introns are spliced out (snRNP complexes recognise and bind splicing sequences)
• 5’GPPP cap is added
• 3’poly A tail is added
• mRNA has to be translocated to cytosol: then translation
• RNA pol binds through the initiation complex (TATA box) to the promotor
• regulator proteins bind distantly to the DNA and influence RNA pol. activity

Question 3

5’GPPP cap. function, production, name

Answer 3

5’cap of 7-methyl guanosine triphosphate. three enzymes needed:
- RNA triphophatase
- guanylyltransferase
- methyltransferase.
only function when they are attached to RNA polymerase II.
It protects RNA from degradation and is needed for transport of mRNA out of the nucleus and guiding them to ribosome.

Question 4

Poly A tail, synthesised by, function

Answer 4

AAUAA. poly-a-polymerase (PAP) synthesises this tail in a template independent manner. eventually more proteins bind to the polyA tail.

function: protects RNA from degradation, needed for transport out of nucleus.

Question 5

Q-PCR

Answer 5

fluorescent probes are attached to RNA. this way the increase in fluorescence/RNA levels can be measured real time. There is a threshold to detect fluorescence. more cycles: less RNA, less cycles: more RNA.

Question 6

cis-elements

Answer 6

a regulatory element/DNA seq that influences expression of a gene on the same chromosome/molecule

Question 7

trans-acting factors (elements)

Answer 7

is present on a different chromosome and influences expression of a gene via the protein it encodes.

Question 8

how to visualise DNA binding proteins

Answer 8

• chemical/enzymatic degradation: DNA bound by protein is better protected. Add chemical: unbound DNA will degrade.
• mobility shift assays: DNA fragments incubated with protein mixes –> if protein binds to DNA –> change of electrophoretic properties –> DNA will move slower through the gel.

Question 9

biogenesis of miRNA

Answer 9

-RNA pol II transcribes miRNA gene
- pri-miRNA is formed, and consists of loop structure of ds RNA
- Drosha cuts away polY and 5’cap- –> pre-miRNa
- translocation out of nucleus
- Dicer cuts it into miRNA
- miRNA is loaded onto RISC complex
- one strand gets displaced, the other binds to target RNA
- target RNA gets degraded/inhibited