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Biochem Part III > Lecture 37: RNA Structure, Synthesis, Processing II > Flashcards

Lecture 37: RNA Structure, Synthesis, Processing II Flashcards

1
Q

Eukaryotic mRNA processing modifications

A
  1. 5’ cap
  2. 3’ polyA tail
  3. Intron splicing
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2
Q

mRNA processing locations

A

Eukaryotic mRNA has to be processed in the nucleus before export to cytoplasm, where translation occurs

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3
Q

Prokaryotic mRNA processing

A

Prokaryotes don’t do mRNA processing! Replication, transcription, translation are simultaneous and in the same place.

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4
Q

5’ cap

A
  • GTP added backwards to 5’ end of mRNA
  • Only 5’-5’ tri-Pi linkage
  • Catalyzed by guanylyl transferase
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5
Q

5’ cap addition process

A
  1. γ Pi released from RNA
  2. PPi released from GTP
  3. Condensation
  4. N7 methylation in nucleus
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6
Q

5’ cap functions

A
  1. Protect vs nuclease digestion
  2. Scaffold for protein binding
  3. Efficient translation
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7
Q

3’ polyadenylation tail

A
  • Protects 3’ end from degradation (stabilizes mRNA)
  • Added during transcription termination
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8
Q

3’ polyA addition process

A

Eukary. transcription termination:
1. RNA poly puts AAUAAA cleavage signal to 3’ end
2. Specific endonuclease cleaves downstream
3. PolyA polymerase adds A’s w/ ATP
4. PolyA binding proteins associate

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9
Q

Intron splicing

A
  • Uses small nuclear ribonuclear proteins (snRNPs)
  • Uses unique 2’-5’ P-diester branch bond
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10
Q

Alternative splicing

A

Multiple proteins can be encoded in 1 gene; final expression depends on how transcript is spliced

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11
Q

snRNPs

A

Small nuclear ribonuclear proteins = snRNA + proteins (U1-U6)
- Ribozyme (RNA w/ enzymatic activity)

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12
Q

Intron structure

A

All introns start GU, end AG; consensus seq. at 3’ and 5’ exon/intron borders

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13
Q

Intron branch point

A

A located in pyrimidine rich seq., ~50 bases from intron 3’ end

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14
Q

snRNP roles

A

U1: binds 5’ splice site
U2: binds branch site (catalytic center)
U5: binds 3’ splice site, loops over to 5’ site
U4: temporary U6 catalytic center masking
U6: Catalyzes splicing

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15
Q

How does U1 bind the 5’ splice site?

A

U1 RNA is complementary to the 5’ splice site; many U1 families exist

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16
Q

Spliceosome assembly process

A
  1. U1 binds 5’ end, U2 binds branch site
  2. RNA folds, U4/5/6 complex added
  3. U1/U4 leave; U5 holds splice ends together
  4. U2/U6 exert catalytic activity
17
Q

U2/U6 catalytic complex activity

A

U2/U6 ribozyme have complementary RNA to catalytic site
- Catalyze 2 transesterifications:
1. 2’ OH of A branch point attacks 5’ splice site
2. 3’ OH of exon 1 attacks 3’ splice site

Intron leaves as lariat (loop)

18
Q

Beta thalassemia major, splicing pathology

A

Point mutations can create new splice sites (req. close enough to same branch point), introducing new stop codons e.g. no functional beta expression

19
Q

Exon/Intron Splicing Enhancers vs Silencers

A

Enhancers bind SR proteins (Ser+Arg rich, favor splicing i.e. exon spliced in)
Silencers bind hnRNPs, inhib. splicing i.e. exon not spliced in
E.g. CD44 v5 exon 10 in cancer

20
Q

tRNA

A
  • Same in prokary/eukary.
  • Features post-transcriptionally modified bases, not just AGCU
  • Clover leaf structure w/ acceptor stem, anti-codon region
21
Q

tRNA acceptor stem

A

Site of AA charging by synthetases

22
Q

tRNA anti codon region

A

Interacts w/ mRNA

23
Q

Bacterial tRNA features

A
  • Multimeric (processed to monomer precursors)
  • Cleaved 5’ and 3’ by RNAses
  • Modified bases
  • 3’ CCA added by tRNA nucleotidyl transferase
24
Q

Prokaryote tRNA transcript processing

A
  1. 5’ end cleavage
  2. 3’ end cleavage
  3. Base modification
  4. CCA added to 3’ by nucleotidyl transferase
25
Q

Eukaryotic tRNA features

A
  • Monomers
  • Req. removal of intron near anticodon
  • Also 3’ CCA, base modifications
26
Q

Prokaryotic ribosome subunits

A

50S + 30S = 70S

27
Q

Eukaryotic ribosome subunits

A

60S + 40S = 80S

28
Q

Ribosomal subunit composition

A

rRNA + proteins; medium rRNA forms smaller subunit

29
Q

Prokaryotic rRNA to subunit

A

50S = 23S, 5S rRNA
30S = 16S rRNA

30
Q

Eukaryotic rRNA to subunit

A

60S = 28S, 5.8S rRNA
40S = 18S rRNA

31
Q

Prokaryotic rRNA synthesis

A

All rRNAs synth. 1 large precursor then cleaved

32
Q

Eukaryotic rRNA synthesis

A

Poly I in nucleolus
Synth. as monomeric precursors from concatenated genes (moderately repetitive seq’s, many copies of gene-promoter joined together)
Creates 45S rRNA precursor which self-splices; subunits self-assemble in nucleolus -> separate until active in cytoplasm

33
Q

Negative feedback of eukaryotic rRNA transcription

A

Excess 5S rRNA inhibits further transcription

Biochem Part III (14 decks)

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