Biol 304 Discussion 9

27. How many different RNA species could theoretically be produced from a transcription unit that contains 3 exons & 2 introns?

4 -must stay in order -can't have lone exon

37. Do all messages have 5' and 3' UTRs? Do all eukaryotic messages have 5' and 3' UTRs?

No; No

13. Do all genes in eukaryotes have introns?

Nope

18. Are introns removed in order? Are exons spliced in order?

Not always; yes

6. What is the role of the AAUAAA sequence once it becomes transcribed into the RNA chain?

signals termination 1. recognizes CPSF 2. recruits CSTF 3. recruits endonuclease CFI and CFII

21. What's the difference between snRNA & snRNP?

snRNP = snRNA + proteins

33. What enzyme removes A nucleotides from the Poly(A) tail?

polynuceases AKA deadenylase

31. What is the structure called when many ribosomes are translating one message?

polysome

41. What is the general function of microRNAs?

prevent translation

3. Every time a nucleotide is added to a growing RNA, what part of the new NT is lost? What is an exception to this?

pyro phosphate is lost (PPi) exempt = 5' cap

11. What is the function of Cleavage and Polyadenylation Specificity Factor (CPSF)?

recognizes AAUAAA sequence

20. What is RNP?

ribonucleoprotein

16. Does splicing require a particular shape or a particular sequence?

sequence

32. What are 2 functions of PABP?

-stabilizes poly(a) tail -circularizes whole RNA

34. How many common pathways for degradation of mRNA are present in eukaryotes? Are they sequence specific? What does it mean if they are not sequence specific?

2; no; either mechanism

1. What is so unusual about the 5' cap found on eukaryotic mRNA? What is special about its linkage?

5'-5' linkage (backward) doesn't lose a PPi

22. Among different snRNP "snurps," do they contain similar or different proteins?

8 common 12 unique 1 RNA

% of human genes that are differentially spliced

90%

12. What is the function of Cleavage Stimulatory Factor (CstF)?

Binds CPSF Recruits endonucleases

6 complexes formed during spliceosome assembly:

E A B1 B2 C1 C2

24. What are the components of the spliceosome?

E A B1 B2 C1 C2 snurps snRNA RNA aux factors

5. How does the chain termination differ between prokaryotes and eukaryotes?

Euk: no strict termination Prok: loop & run of U's & rho-dependent (intrinsic)

Spliceosome Assembly

SR protein - contain domains rich with serine and arginine • BBP - Branch binding protein • U2AF - heterodimer of 65 kDa and 35 kDa protein • 65kDa - contacts a 12 prymidine tract • 35kDa - contacts the AG dinucleotide at the 3′ splice site (acceptor site)

19. What molecules catalyze nucleophilic attacks?

U2/U6

15. Can some introns have AU instead of a GU sequence at the donor site? Would that site be recognized by the usual splicing factors?

Yes: U12 Won't recognize it

8. Does the transcript end after, at, or before the AAUAAA site?

after

10. What is the function of pol(a) polymerase or PAP?

binds 3' end, adds long chain of adenines

40. Where are microRNAs found in the genome?

everywhere

23. What's a spliceosome?

complex assembled on pre-mRNA for excision & splicing

17. What is the purpose of the splicing complex?

excise introns splice exons

39. Can degradation occur without the removal of poly(A) tail?

deadenylation independent decapping

38. What is the name of the multicomplex structure in bacteria that is responsible for RNA degradation and what is the analogous structure in eukaryotes?

degradosome; exosome

36. What is a sequence that decreases the stability of mRNA called? What type of molecule is it?

destabilizing sequence; mRNA

43. What happens if a miRNA is fully complementary/partially complementary to the target mRNA?

direct degradation; inhibit translation

45. Why is turning genes off by RNAi easier than creating mutations?

doesn't affect DNA

14. Which elements in introns are conserved?

donor sequence branch site acceptor sequence polypyrimidine tract

25. What are the important sequences in snRNA?

donor sequence branch site acceptor sequence polypyrimidine tract

7. What type of nuclease makes a cut after the AAUAAA site?

endonuclease

9. What is the function of CFI and CFII?

endonuclease cleaves after poly(a) signal

28. Which would you expect to have a longer half-life, a prokaryotic or eukaryotic mRNA, and why?

eukaryotic b/c of g-cap and poly(a) tail

4. What adds the cap?

guanylyl transferase

44. If a gene is transcribed into miRNA and then the miRNA is internally complementary to itself, so it forms a hairpin, what type of repeat has to be found in the gene?

inverted not direct b/c not complementary

2. What are other modifications at the 5' end besides the addition of the G nucleotide that contributes to the cap structure?

methylation @ 5' cap

30. What is the general half life of a bacterial RNA? What can extend or decrease the life span of the message?

minutes; ribosomes

29. Why is RNA less stable than DNA?

more nucleases

46. What might long (+26 base pairs) dsRNA trigger in eukaryotes?

non-specific degradation of-target effects

35. What type of mRNA would you expect to be degraded in eukaryotes?

non-translated mRNAs

SPLICEOSOME ASSEMBLY The spliceosome complex is a large group of proteins and specialized _______________________, which is used to excise the intron sequences and properly join the exons from a pre-mRNA transcript. The snurps are proteins bound to _________, and play an important role in the proper recognition of intron sequences. The spliceosome complex goes through several stages in order to splice the transcript; energy is required in the form of ATP in order for the snurps to properly bind and for the unwinding of certain regions of double-stranded RNA that form, but the _______________________ steps that cleave the RNA require no energy input. Splicing is an important event for eukaryotic gene expression, and the complexity of the spliceosome reflects that fact: each snurp is generally composed of a small U RNA, approximately ____ common core proteins and approximately ______ proteins unique to each snurp. The entire spliceosome assembly is larger than ribosome.

snurps (small nuclear ribonucleoproteins); snRNA; transesterification (nucleophilic attack); 8; 12

26. Do prokaryotes have introns? Do prokaryotes use the spliceosome assembly?

some do; no

MESSAGE STABILITY If RNA transcripts were to last indefinitely, cells would be stuck synthesizing proteins, which no longer may be needed or are already in too great a number. To counteract this issue, transcripts have built-in or modified regulatory elements that allow for their timely degradation. As previously discussed in eukaryotes, the 5' cap and poly(a) tail are modifications, which prevent the transcript from being targeted by nucleases. Eukaryotes also have 5' & 3' UTR's that contain sequences to stabilize or destabilize the transcript. Prokaryotes do not have 5' or 3' modifications to their RNA, although they may have __________ regions that form on the mRNA and prevent degradation. A primary mechanism for their stability comes from the unique prokaryotic ___________, a structure that arises due to the lack of a nuclear envelope. Transcription & translation take place in the same location & at the same time; as the mRNA strand is synthesized, several ribosomes can attach before the transcript has ever completed. A high density of _____________ on the mRNA strand will prevent degradation of the transcript, & when gaps open up the strand can be cut. This process often leaves prokaryotic transcripts susceptible to destruction more quickly; while eukaryotic mRNA may have a half-life ranging from _____________, in prokaryotes it is generally __________.

stem-loop; polysome; ribosomes; hours to days; three to ninety minutes

42. What factors that control gene expression are microRNAs affecting?

translation and message stability

RNA PROCESSING In eukaryotes, pre-RNA transcripts are modified in several ways before they can be used. This is important for pre-mRNA, as it must be modified to prevent its degradation & allow for its proper usage during __________. During transcription, _____________ (which associates with the phosphorylated CTD tail of RNAP II) attaches a special cap to the 5' end of the growing transcript; this cap is a methylated __________ bound backwards __________, with a special ___________ linkage. RNAP continues down the DNA template until it transcribes the sequence 3'-TTATTT-5' into AAUAAA. The inclusion of these bases in an RNA chain serves as a recognition site for the RNA binding proteins that cleave the growing chain and attach a poly(a) tail to the end of the chain. One of these proteins is _________________, which binds to the newly cleaved free 3' end of the transcript, adding a long chain of adenines which can be bound by ____________________. The presence of PABP - relying on a poly(a) tail of at least _________________ - confers stability to the new RNA message.

translation; guanylyl transferase; guanine; 5' to 5'; triphosphate; poly(a) polymerase (PAP); poly(a) binding protein (PABP); 10A nucleotides