MB 2 Transcription

• Transcription and processing of eukaryotic genes

  Models explaining the mechanisms of processing of eukaryotic mRNA during transcription initiation, as well as 3' end processing and transcription termination
• Main components of a transcription initiation complex


  • RNA polymerase II, promoters, general transcription factors
• Order in which transcription is initiated in eukaryotes

  Transcription initiation, elongation, termination
• Types of cells in the body

  • Fat, skin, nerve, blood, osteocyte
• Human DNA is mostly identical in every cell
• Central dogma


  DNA -> RNA -> Protein
• Eukaryotes store DNA in their nucleus
• DNA relays information from the nucleus to the cytoplasm via RNA
• Chromosomes and DNA


  • Chromosomal DNA is wrapped around nucleosomes, only during cell division do x-shaped chromosomes form
• DNA structure


  1. type double helix, complementary base pairs, DNA replication
• RNA structure


  Single stranded, can form Watson-Crick base pairs
• Transcription


  DNA base information is copied into RNA, does not need a primer, transcription proceeds at 33-50 nt/sec, transcription errs 1 in every 10,000 nucleotides
• Types of RNA in eukaryotes


  • Ribosomal RNA, mRNA, snRNA, eRNA, lincRNA, tRNA, catalytic RNA
• Eukaryotic RNA polymerases


  Pol I, Pol II, Pol III, each transcribes a defined set of genes
• Promoter and transcript end signal


  Promoter is the start, TES is the stop
• mRNA processing


  Capping, splicing, 3' end processing (cleavage and polyadenylation)
• Not all genes are transcribed in every cell
• Levels of gene expression control

  Transcriptional control, RNA processing, RNA transport and localisation, translational control, mRNA degradation control, protein activity control
• In eukaryotes, one transcription unit usually encodes for one gene
• Stages of Pol II transcription and RNA processing


  Initiation, elongation, termination, capping, splicing, 3' end processing
• DNA binding proteins


  Recognise 6-8 base pairs, weak interactions in combination of 10-20 cause tight binding
• Protein binding can bend DNA
• Consensus sequences


  Sequence logos indicate frequency of nucleotides in a binding sequence
• Pol II promoters


  • TATA box, BRE, DPE, Inr
• Pol II promoter binding

  Requires DNA opening, interplay of general transcription factors, activators and DNA torsions
• Pre-initiation complex (PIC) assembly


  TBP kinks DNA, TFIID binds Inr and DPE, TFIIA displaces Taf1 so TBP can bind, TFIIB recruits Pol II-TFIIF complex, TFIIH translocates and opens promoter
• Transcription initiation


  Tightly regulated to prevent unscheduled transcription
• Pre-Initiation complex (PIC) assembly

  • TFIID: TBP and 13 TFIID-associated factors (TAFs)
  • TFIID binds INR and DPE, delivering TBP to DNA
  • TBP-DNA binding is inhibited by Taf1 which mimics DNA
  • TFIIA is recruited and displaces Taf1, so that TBP can bind DNA
  • Distance between TBP and TFIID is ~32 bp regardless of sequence
• From PIC to open promoter

  1. TFIID establishes sequence specificity and controls TBP binding

  2. TBP kinks DNA

  3. TFIIB is recruited to TBP-TFIIA dimer and binds BRE

  4. TFIIB recruits Pol II-TFIIF complex

  5. TFIIH XPB translocates and helps open up DNA

  6. Additional bending introduced by TFIIE and TFIIH

  7. TFIIB and XPB increase bending and positive supercoiling, allowing opening up of DNA in active site

  8. TFIID, TFIIB, Pol II, TFIIF are recruited: Point of no return
• The core promoter sequence is not present in all promoters
• Transcription by RNA polymerase II
• Transcription as seen under the electron microscope
• Transcription elongation and splicing

  1. Initiation

  2. Elongation

  3. Termination

  4. Capping

  5. Splicing

  6. 3' end processing: Cleavage and polyadenylation
• RNA Pol II C-terminal domain (the CTD)


  • 52 heptad sequence repeats with consensus Y1S2P3T4S5P6S7
  • TFIIH CDK7 phosphorylates S5P, abortive inititation overcome
• Phospho-CTD as sorting-tool and landing pad


  • RNA processing is coupled to transcription through phospho-CTD-specific binding
  • Elongation factors can bind phospho-CTD to prevent premature termination of stalled polymerases as well as facilitate passage through nucleosomes
  • Kinases change CTD phosphorylation patterns co-transcriptionally
• Transcription stages


  1. Initiation

  2. Elongation

  3. Termination
• RNA capping


  Three different enzymes in yeast, two different enzymes in humans
• Protein coding sequences are interrupted by introns
• Splicing involves dynamic RNA base-pairing and RNA-mediated catalysis
• 5' and 3' untranslated regions


  • 5' UTR
  • 3' UTR