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BIOL200 > RR8: RNA Processing I > Flashcards

RR8: RNA Processing I Flashcards

1
Q

How many RNA polymerases do eukaryotes possess?

A

3.
RNA polymerase I
RNA polymerase II
RNA polymerase III

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2
Q

What are the common features of the 3 different RNA polymerases?

A
  • They are multimeric protein complexes (have a lot of subunits)
  • The subunits are similar to bacterial RNA polymerase
  • Their subunits are all more or less essential
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3
Q

What’s something that’s different in RNA polymerase II?

A

CTD. Carboxy-terminal domain.
It’s on a large subunit of RNA pol 2

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4
Q

What is the CTD made of?

A

It’s made of 52 heptapeptide repeats of YSPTSPS (7).

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5
Q

Are the repeats important in CTD?

A

Yes, if we take out the repeats, the organism dies, they’re essential for life.

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6
Q

What does the phosphorylation of the CTD?

A

TFIIH

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7
Q

Which residue gets phosphorylated by TFIIH (it has a protein kinase)?

A

The 5 S on the YSPTSPS repeat.

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8
Q

Which other residue gets phosphorylated?

A

S 2. It’s the second phosphorylation but it’s done by a protein kinase, CDK9, not TFIIH. It happens during elongation. That phosphorylation switches initiation to elongation.

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9
Q

After the initiation of transcription, RNA pol 2 stops after 100 nucleotides. Why?

A

The phosphorylation of the 5 S done by TFIIH gives rise to specific structures on the heptapeptide repeats that are required for the merging of the 5’ end of pre-mRNA.
Then, the phosphorylation of S5 next to the CTD recruits a capping enzyme that will add a 7 methyl guanosine cap to the 5’ end of the pre-mRNA.
RNA pol 2 pauses to give time to protect the 5’ end by capping it.

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10
Q

What does RNA polymerase 2 need to do in order to initiate transcription?

A

It has to leave all the other transcription factors and the promoter. It clears.

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11
Q

What are the roles of the CAP?

A

Recognize the 5’ end and add the 7’ methylguanylate cap to the 5’ end.
Protect the 5’ end of pre-mRNA from exoribonucleases.
Facilitate nuclear export.
Recruiting factors for efficient translation.

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12
Q

What protects the 5’ end of pre-mRNA during the initiation of transcription?

A

The capping enzyme putting the 7’ methylguanosine cap.
The 2’ hydroxyl on the second nucleotide getting phosphorylated at the same time as the cap is put.

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13
Q

What is NELF?

A

Negative elongation factor.
Protein that binds to RNA pol 2 when it leaves the pre-initiation complex.
It blocks elongation.
Blocks NTPs from getting into the catalytic site, so RNA pol 2 stops elongation.

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14
Q

What is DSIF?

A

DRB Sensitive Inhibitor Factor.
Protein associated with NELF.
Enhance elongation.

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15
Q

What is CDK9?

A

It’s a cyclin-dependent kinase.
It phosphorylates
- S 2 on the heptapeptide repeats of CTD.
- DSIF
- NELF
Coupled with cyclin T.
It recognizes the pause done by RNA pol 2 and phosphorylate S 2 on the CTD.
Forces DSIF to close the clamp, so plays a critical role in changing the stop to an elongation complex.

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16
Q

What happens directly after the phosphorylation of S2 on the heptapeptide repeat of CTD?

A

NELF leaves and PAF takes its place to make sure NELF can’t come back.

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17
Q

What happens to DSIF when it gets phosphorylated by CDK9?

A

Before, it was a negative elongation factor.
After phosphorylation, it closes the clamp down, enhancing transcription.
Then, the other elongation promoting factors associate with the complex to pursue elongation.

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18
Q

Why are serine 2 (s2 phosphorylated by cdk9) important?

A

Recruit additional proteins:
- splicing factors
- polyadenylation factors
- export factors.
So they can modify the pre-mRNA as it’s getting processed.

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19
Q

How to we get from pre-mRNA to mRNA?

A

Splicing.

20
Q

What is splicing?

A

Taking out introns from the pre-mRNA transcript.

21
Q

Do every organism have introns?

A

No. Bacteria don’t have introns. Humans have huge introns.

22
Q

Are introns important even if they’re spliced?

A

Yes, they’re important. They can encode regulatory information like cis-acting elements and enhancers.

23
Q

How were introns discovered?

A

They realized the difference in size between mRNA and gene size.
Introns were visualized by hybridization experiments, they saw that intron sequences in the DNA loop out.

24
Q

What is special about the border sequences of introns?

A

They’re highly conserved.
5’ end of the intron: G U
3’ end of the intron: A G
It’s very conserved.
There’s also the pyrimidine-rich region that’s conserved near the 3’ end.
There’s a branch point A that’s conserved in the middle of an intron.

25
Q

Why is it important for intron sequences to be conserved?

A

Because U1 snRNA has to base pair to the RNA, so if there’s a small change in the nucleotide sequence, it can block splicing.

26
Q

What are snRNAs?

A

Small nuclear RNAs.
They’re essential for spliceosome functionality.

27
Q

What is a spliceosome?

A

Ribonucleoprotein complex: consists of 5 snRNPs (small ribonucleoprotein particles).
Removes introns from pre-mRNA to generate mature mRNA.
Performs splicing.

28
Q

What are the snRNAs?

A

Small nuclear RNA:
- U1
- U2
- U4
- U5
- U6

29
Q

What is snRNA used for?

A

Essential for splicing.
They’re associated with proteins called small nuclear ribonucleoproteins (snRNPs)

30
Q

Which snRNAs interacts with the intron 5’ border?

A

U1 snRNA with the intron 5’ border.

31
Q

Which snRNA interacts with the branch point region A?

A

U2 snRNA with the branch point region A.
The actual A base is not connected to anything, so it bulges out. It’s the region around it that’s connected to U2 snRNA.

32
Q

What generates snRNAs?

A

RNA polymerase 2 generates U1, U2, U4 and U5.
U6 is NOT generated by RNA pol 2.

33
Q

What’s the essential condition for splicing to happen?

A

Watson Crick base pairing.
As long as U1 snRNA can interact with the nucleotide sequence with the Watson Crick base pair at the 5’ end of the intron, then splicing can take place.
U2 snRNA also has to Watson Crick base pair with the nucleotides around the adenosine branch point.

34
Q

How does the Watson Crick base pair of U2 snRNA and the nucleotides around the adenosine branch point affect the splicing of introns?

A

It’s dependent on 2 trans-esterification reactions.
First trans-esterification: Hydroxyl group on the 2’ position of the bulged-out A carries a nucleophilic attack on the phosphorus of the upstream exon. (2’ - 5’ linkage and a new RNA lariat structure (like a lasso))
Second trans-esterification: the free hydroxyl group on the 3’ end of the upstream exon carries a nucleophilic attack on the phosphorus of the 5’ end of the downstream exon.

35
Q

If we were to radiolabel RNA substrates with probes on a gel, where would the lariat structures be?

A

The lariat structures act like a high molecular weight molecule, while the exon-intron molecule and the spliced RNA are both lower weight on the label.

36
Q

What are the steps of splicing with the spliceosome cycle?

A
  1. snRNPs assemble on the intron:
    - U1 snRNP + U1 snRNA interacts with the 5’ end of the intron
    - U2 snRNP + U2 snRNA base pair the nucleotides next to the A branch point
    - U4, U5, U6 snRNPs + their snRNAs recognize the complex and join
  2. Reajustments of RNA-RNA interactions between pre-mRNA and snRNA
  3. U1, U4 snRNPs + their snRNAs leave the spliceosome
  4. U2, U5, U6 snRNPs + their snRNAs form the active spliceosome
  5. During the active splicesome, the trans-esterification reactions occur (very fast)
  6. After the trans-esterifications by U2 snRNA, we get 2 exons together linked by a phosphodiester bond.
  7. U2, U5, U6 snRNPs + their snRNAs leave
  8. We’re left with the lariat intron
37
Q

How do we deal with the lariat intron after it’s been spliced from the mRNA?

A

Because it’s a 5’ - 2’ linkage it’s hard to get rid of it using regular degrading enzymes.
We use a debranching enzyme that will linearize the intron.
Once it’s linearized, it can be degraded by exo and endoribonucleases.

38
Q

What are self-splicing introns?

A

Introns that don’t need proteins to be spliced. They can be removed by the RNA themselves.

39
Q

What does the fact that RNA can spliced itself without the need of any protein can suggest about RNA?

A

It can mean that RNA has a catalytic function.
Nucleic acids can be catalytic, so they can do an enzymatic reaction without proteins.
Maybe RNA came first, then proteins and after DNA came after.

40
Q

How did we find out that RNA can splice itself?

A

A linear form of RNA has the be spliced to form circular RNA.
The splicing reaction needs magnesium.
Then, we put a proteinase in the mix that will eat all the proteins.
As long as magnesium was there, the splicing still happened, so the proteins were actually not needed for splicing to happen.

41
Q

Does self-splicing intron happen to every mRNA?

A

No. Self-splicing is the exception, not the rule.

42
Q

What are the 2 types of self-splicing introns?

A

Group 1
Group 2

43
Q

What complex does most of the splicing reactions (if self-splicing is less frequent)?

A

The splicesome. A ribonucleoprotein complex formed by snRNPs.

44
Q

Is self-splicing as efficient as the spliceosome?

A

No. The proteins in the spliceosome make the splicing much more efficient.

45
Q

What are the differences between group 1 and group 2 self-splicing introns?

A

group 2 introns are only present in mitochondria and chloroplast genes

46
Q

How does self-splicing occur?

A

They fold into secondary structures, so the residues are close so it can give rise to trans-esterifications.

BIOL200 (16 decks)

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