mcb 121 topic 6

• Regulation of gene expression is the major factor leading to phenotypic differences between species and cell types
• Control of transcription initiation is one of the most important steps of regulation for Type II genes (protein-coding genes)
• Type II genes have binding sites for general (basal) transcription factors, which bring RNA Pol II to the promoter
• Eukaryotic RNA Polymerases


  • Bacteria have 1 RNA polymerase, eukaryotes have three different RNA Polymerases (RNA Pol I, RNA Pol II, and RNA Pol III)
  • All RNA polymerases are related at the sequence and structural level
  • Eukaryotic RNA polymerases have more subunits than the bacterial RNA Polymerase
  • Transcription is more complicated in eukaryotes because DNA is compacted into chromatin
• Types of RNA Polymerases in Eukaryotes

  • RNA Polymerase I transcribes Type I genes: rRNAs (busiest polymerase)
  • RNA Polymerase II transcribes Type II genes: All protein-coding mRNAs, many noncoding RNAs (e.g. miRNAs)
  • RNA Polymerase III transcribes Type III genes: 5S rRNA, tRNAs
• Similarities between RNA Polymerases


  • All contain 10 common subunits
  • All use a conserved catalytic mechanism
• Differences between RNA Polymerases


  • Different RNA Polymerases recognize different promoter sequences
  • Different RNA Polymerases have different regulatory subunits and control mechanisms
• Promoter Architecture - Type I Genes


  • RNA Polymerase I transcribes only one gene, the rRNA precursor
  • The upstream control element (UCE) is bound by transcription factors that are required to recruit RNA Polymerase I
• Promoter Architecture - Type III Genes


  The Internal Control Region (ICR) is bound by transcription factors that recruit RNA Pol III
• Promoter Architecture - Type II Genes


  • Consensus sequence of the core promoter includes: TATA Box, BRE, Inr, DPE
  • The basal transcription factors bind to the consensus sequences in the core promoter
• RNA Polymerase II Transcription Mechanisms

  1. Formation of closed Pre-initiation Complex (PIC): TFIID binds to TATA box, recruitment of TFIIA, TFIIB, RNA Pol II, TFIIF, TFIIE, TFIIH

  2. Formation of the "Open PIC" (transcription bubble): Unwinding of DNA at TSS by TFIIH helicase activity

  3. Phosphorylation of the CTD during initiation: Phosphorylation of Ser5 and Ser7 by TFIIH kinase

  4. Elongation: RNA Pol II pauses, PTEF-b required to transition to elongation, phosphorylation of Ser2

  5. Termination and dephosphorylation of the CTD: Cleavage of nascent transcript, RNA Pol II falls off, CTD dephosphorylated
• Chromatin structure


  • Euchromatin (11nm fiber): less condensed, transcriptionally active
  • Heterochromatin (30nm fiber): more condensed, transcriptionally silent
• Histone modifications


  • Acetylation: Reduces affinity of histone tails for DNA, catalyzed by HATs and HDACs
  • Methylation: Tails remain positively charged, chromatin closed, catalyzed by methylases and demethylases
• ChIP-seq is a method to determine the locations that a specific protein is bound to DNA on a genome-wide scale
• Nucleosomes need to be moved away from the TSS so that RNA Polymerase and associated factors can bind
• CUT&RUN and CUT&Tag are more modern versions of ChIP-seq that produce more precise results with less material