Chapter 6 - Transcription

• RNA Polymerases: Pol I, Pol II, Pol III; Don't require primers, 1 mistake in 10^4 nucleotides; Modest repair activity
• Pol I - 5.8 S, 18 S, 28 S rRNA
• Pol II - mRNA, snoRNA, snRNA
• Pol III - tRNA, 5S rRNA, snRNA, other small RNAs
• RNA Pol II needs general transcription factors to bind to the promoter and initiate transcription
• RNA Polymerases:

  • Pol I: synthesizes 5.8 S, 18 S, 28 S rRNA
  • Pol II: synthesizes mRNA, snoRNA, snRNA
  • Pol III: synthesizes tRNA, 5S rRNA, snRNA, and other small RNAs
  • RNA Polymerases don't require primers
  • 1 mistake in 10^4 nucleotides (DNA has 1 mistake in 10^7)
  • Modest repair activity
• Transcription Initiation:

  • RNA Polymerase II needs general TF
  • TFIID: TBP subunit binds TATAA
  • TFIIA binds TBP
  • TFIIB binds DNA and TBP
  • TFIIF/RNA Pol II form pre-initiation complex
  • TFIIE is a docking site for H
  • TFIIH has helicase activity, to open up DNA, and phosphorylates Pol II
• Transcriptional Elongation:

  • Phosphodiester bond in RNA
  • Many copies can be made at once
  • Reads 3’ to 5’
  • 20 nt/second
  • Topoisomerase
  • RNA does not remain H bonded to DNA
  • Elongation factors
• Transcriptional Termination:

  • AAUAAA sequence signals termination
  • Pol and mRNA are released
• 5' cap

  • Modified guanine after 25 nt made on pre-mRNA
  • Phosphatase removes 1 phosphate from 5’ end
  • Guanyl transferase - GMP in reverse linkage from 5’ to 5’
  • Methyl transferase adds methyl to 5’
• Roles of 5' cap

  Signals 5' end, Distinguishing mRNAs, Translocation out of nucleus to cytoplasm, Translation, Protection
• 3' Poly-A Tail:

  • Complex on Pol II - CstF and CSPF
  • Transferred to 3' end of pre-mRNA
  • RNA is cleaved from DNA then poly-A polymerase adds 200 A
  • Doesn't need template (not directed by genome)
• Role of 3' Tail?

  Translation, Protection, Nuclear Export
• Intron Spicing

  • 2 Transesterification events
  • 10-100,000 nucleotides
  • 5 RNA molecules (snRNA): U1, U2, U4, U5, U6 ; Less than 200 nt ; Recognize splice sites
  • 3 sites: 5' Splice Site, Branch Point Site (A), 3' Splice Site
  • Over 50 proteins
• Intron Splicing - General Steps:

  1. 2'OH of A attacks 5' splice site
  2. 3'OH of Exon 1 attacks 3' splice site
  3. Exons joined and Intron excised
• snRNA and a protein combine to create Small nuclear ribonucleoproteins (snRNPs)
• The spliceosome is a complex of proteins that removes introns from pre-mRNA

  • All snRNPs in a complex with the mRNA to be spliced
  • RNA Pol II carries many components of the spliceosome
• Spliceosome process:

  1. BBP binds to A in branch point site
  2. U2 displaces BBP as a double check step
  3. U1 binds 5' site
  4. U4/U6-U5 Triple complex enters: 2'OH of A attacks bond between Exon 1 and the G residue (cleaves at GU)
  5. U4 ejected, U6 displaces U1, New bond is formed between A and G forming a loop structure (First transesterification Event)
  6. U5 regognizes 3' splice site
  7. 3' OH end of Exon 1 attacks 3' splice site / cleaves bond between G and E2
  8. New bond is formed between E1 and E2 (Second Transesterification Event)
• Why does U2 displace BBP?

  First double checking step
• Why does U6 displace U1?

  Second double checking step
• First Transesterficication Event in Intron Splicing?

  2' OH of A attacks bonds between Exon 1 and G residue; U4 is ejected, U6 displaces U1; New bond formed between A and G forming loop structure
• Second Transesterification Event in Intron Splicing?

  3'OH at end of Exon 1 attacks 3' splice site and cleaves bond between G and Exon 2; New bond is formed between Exon 1 and Exon 2; snRNPs recycled
• What is a transesterification event?

  Breaking of 1 phosphodiester bond and creating another phosphodiester bond
• Fidelity of Splicing

  • BBP and U2 snRNP - Branch point site check
  • U1 and U6 snRNPs - 5' splice site check
  • Exon Definition Hypothesis - Exons ~150nt and introns vary, SR proteins recruit U1 and U2
• Importance of Splicing:

  1. Regulatory regions - control gene expression/Enhancers
  2. snoRNA - guide RNAs
  3. Alternative splicing
• What is snoRNA?

  • small nucleolar RNA
  • made by RNA Polymerase II
  • Guide RNAs
  • Bring modifying enzymes to mRNA molecules
  • Can be coded for in DNA introns
• rRNA Production:

  • 80% of total RNA
  • 200 rRNA genes per haploid cell
  • No 5' cap/3' poly-a tail
  • Ribosomes
  • 4 types: 5S, 5.8S, 18S, 28S
• What are the 4 types of rRNA?

  5S, 5.8S, 18S, 28S
• 5S rRNA is made by Pol III and is not modified
• 5.8S, 18S, 28S rRNA

  • made by Pol I
  • Pre-cursor 13,000 nt
  • Methylations of 2'OH
  • Isomerization of uridine
• Upstream to Downstream on template DNA strand: Enhancer (Activators/Repressors), Promoter (TATAA, General TF, -25), (-1) Point of Interest (+1), Gene (RNA Polymerase), Terminator Sequence (TTATT)