Lecture 23: Eukaryotic Transcription, RNA polymerases, m7g capping, polyadenylation

Question 1

euchromatin

Answer 1

• transcriptionally active
• associated with nuclear pore

Question 2

heterochromatin

Answer 2

• transcriptionally inactive

Question 3

Eukaryotic RNA polymerase (list)

Answer 3

• RNA polymerase I - synthesizes ribosomal RNA 14 subunits
• RNA polymerase II - synthesizes mRNA & snRNAs 12 subunits
• RNA polymerase III - synthesizes small stable RNAs -
• 5S rRNA, tRNA, snRNA (small nuclear RNA), telomerase RNA
• 16 subunits

Question 4

Eukaryotic RNA polymerases (general structure)

Answer 4

• all contain 5 subunits (homologous ot prokaryotic
• largest subunit is Beta prime
  
  • template
  • RNA 7 AA repeat at c-terminal domain (CTD)
    
    • YSPTSPS
• second largest subunit is beta
  
  • synthesis

Question 5

Proof of 3 classes of RNA polymerase

Answer 5

ion exchange chromatography

Question 6

RNA polymerase inhibitors

Answer 6

• Rifampicin: e. coli, human mitoch
• alpha-Amanitin: human I (large amounts), human II (small amounts), human III (medium)

Question 7

rRNA gene transcription

Answer 7

• in tandem array
• non-transcribed spacer between each gene
• RNAP I - highly processive
  
  • recognizes distinct promoters, transcription factors and tata box binding protein
  • UCE (upstream control element) recruits SL1 and UBF (upstream binding factor)
  • these trans factors help bind polymerase
  • RNAP I and III - coordinated
    
    • RNAP I forms 45S pre-rRNA -
    • then split into 20S and 32S -
    • they become 18S and 28S+5.8S
    • 28S+5.8S is combined with 5S from RNAPIII to form 60S ribosomal subunit
    • 18S becomes 40S ribosomal subunit
• in nucleolus - fibrillar structure
• termination of transcription at two large hairpin structures

Question 8

RNA polymerase III (eukaryotes)

Answer 8

• makes tRNA
  
  • have internal promoter sequences
    
    • box A and box B
    • TFIIIC - transcription factor polymerase III: interact with box B and recruit TFIIIB to start site
• makes 5s rRNA
  
  • TFIIIA recognizes dna directly - recruits TFIIIC and TFIIIB
• termination
  
  • run of four As in template
  • like Rho independent termination in bacteria, without requirement for upstream hairpin structure
    *

Question 9

mRNA transcription

Answer 9

1. synthesis of primary transcript
2. modification of 5’ and 3’ ends
3. RNA splicing removes introns. exons are left (coding sequences)

Question 10

RNA polymerase II (Eukaryotes)

Answer 10

• Cis control elements
  
  • eukaryotes:
    
    • more regulatory sequences than eubacteria.
    • can be further from transcription start site
• core promoter: supports basal transcription
• process:
  
  1. TATA binding protein (TBP) and Transcription binding protein associated factors (TAFs) make up the TFIID and bind.
     
     1. TBP interacts antiparallel to sequence in the minor groove
     2. TBP causes bending of DNA
     3. TFIIA hangs off the side of the saddle
  2. then TFIIB binds.
  3. recruits TFIIF, which recruits RNA polymerase and mediator
  4. recruits TFIIE - still a closed complex
  5. TFIIH makes the complex open
  6. as it goes into elongation mode, phosphorylation of CTD causes the loss of the basal transcription factors
  • CTD: heptapeptide (YS*PTS*PS)
    
    • Serine5P: promoter escape, recruits capping machinery
      
      • dephosphorylation causes dissociation of capping machinery
    • Serine2P: elongation
      
      • polyadenylation
      • recruits splicing machinery

Question 11

enhancers

Answer 11

bind activator

Question 12

mediator

Answer 12

co-activator

Question 13

7-methyl G capping (RNA polymerase II)

Answer 13

• phosphorylating the Serine5 recruits capping machinergy
• desphorylating the S5P dissociates capping machinery
• inverted methyl 7 G cap added by 3 enzymes:

1. RNA triphosphatase: removes gamma phosphate off 5’ end of RNA
2. guanylyl transferase: beta phosphate of RNA attacks alpha phosphate of m7G cap
3. methyl transferase: methylates 7 position of m7G

• uses:
  
  • signal export from nucleus
  • initiation of translation
  • stability by protecting from nucleases due to inverted cap structure with phosphate bridge

Question 14

Splicing machinery (RNA polymerase II)

Answer 14

• partially dephosphorylated RNAP II (Serine 2 phosphate): recruits splicing machinery and polyadenylation

Question 15

Polyadenylation (RNA polyermase II)

Answer 15

• Serine2 Phosphate recruits polyadenylation machinery
• RNAPII transcribes beyond polyadenylation signal *AAUAAA)
  
  • over phosphorylation of CTD - dissocates CPSF/CstF
  • CPSF and CstF bind to polyA in RNA - cleavage by CFI and CFII
  • poly A polymerase (a terminal transferase) adds 200-500 As
  • Poly A binding protein added.
• uses:
  
  • singals export from nucleus
  • stability

Question 16

Function of m7G cap and poly-A tail

Answer 16

• provide signals for export from nucleus
• m7G cap - initiation of translation
• stability:
  
  • m7P is inverted resists protease
  • poly A tail resists because specific sequences are avoided.