pp 18

Question 1

What is the basic mechanism of polyadenation?

Answer 1

1. transcription of eukaryotic genes extends beyond the polyadenylation site. Pol II transcribed the polyA addition site
2. Polyadenylation factors find the poly A signal initiating mRNA cleavage
3. cleavage occurs after the AAUAAA polyadenylation signal, PAP (poly A polymerase) syntehsizes the poly A tail, PABP protects tail from degradation

Question 2

What does the mammalian polyadenylation signal consist of?

Answer 2

• AAUAAA motif about 20 nucleotides upstream of a polyadenylation site in pre-mRNA
• followed by 23 or 24 bp later by GU-rich motif and then U rich motif

Question 3

What does polyadenylation involve in mammals?

Answer 3

interaction of several proteins including cleavage and polyadenylation specificity factor, cleavage stimulation factor, cleavage factors 1,2 and poly A polymerase, RNA polymerase II

Question 4

What is the overview of polyadenylation?

Answer 4

1. cleavage of mRNA occurs 20 nucelotides downstrem of AAUAAA motif
2. Initiation of polyadenylation occurs when poly A polymerase slowly adds the first ten or so A’s to the 3’ end of cleavage site
3. After addition of a few A residues, PAP-binding proteins binds and stimulates PAP activity to add around 250 A

Question 5

What does it mean when the poly A tail experiences turnover?

Answer 5

cytoplasm poly A tail of mRNA is shortened, degraded and extended in cytoplasm. When poly A tail is completely degraded the mRNA is rapidly degraded

Question 6

What is the experiment to see the translatability of capping and polyadenylation affecting mRNA?

Answer 6

Introducing various versions of luciferase mRNA into tobacco cells, followd by the amount of mRNA with time and measured amount of activity,
With the cap and poly A tail, expression increases 450 fold. Having the cap and not poly 5 til increases more than having the poly A tail but no cap.

Question 7

What are RNA processign events?

Answer 7

capping, intron splicing and polyadenylation

Question 8

What are the processing events coordinated by?

Answer 8

CTD of RNA polymerase II

Question 9

What happens after the cleavage of the poly A signal sequence?

Answer 9

new 5’ end of RNA still being elongated lacks 5’ cap. Xrn2p exonuclease binds exposed, uncapped end and chases polymerase. Once it catches up, results in the dissociation from the DNA (torpedo mechanism)

Question 10

How does the processing of tRNA happen?

Answer 10

tRNA matured from a primary transcript following cleavage, base modification and base additions

Question 11

How does the processign of rRNA happen?

Answer 11

rRNA are cleaved from larger pre-rRNA transcripts. Maturation of the rRNA involve cleavage of pre-rRNAs and methylation at 2’ OH groups at specific nucleotide residues

Question 12

What are the three rRNAs needed for functional ribosomes in bacterias?

Answer 12

16S, 23S and 5S that arise from a single 30S pre-rRNA

Question 13

What are the three rRNAs needed for functional ribosomes in eukaryotes?

Answer 13

18S, 28S and 5.8S arising from a single 45S pre-rRNA