mcb 121 mt 2

• Transcription


  The process of synthesizing RNA from a DNA template
• RNA Polymerase

  • Catalyzes the formation of RNA
  • Uses one strand of DNA as a template
  • RNA synthesis occurs in the 5' to 3' direction
  • Only a portion of the genome is transcribed in each cell (makes cells different from each other)
• Slides 4-5 shows the coding strand and the template strand. RNA polymerase reads the template strand to produce an RNA that is the same sequence as the coding strand
• RNA Polymerase Enzymatic Activities


  • Polymerase activity (synthesize RNA nucleotides into an RNA polymer)
  • Helicase activity (melts the duplex DNA so that the template strand can be accessed)
  • Dissociate the RNA from the DNA after synthesis. During transcription, only 8-9 RNA nucleotides remain duplexed with the DNA at any moment
• Gene Structure


  • Regulatory elements
  • Transcription Start Site (TSS)
  • Transcription Stop Site
• Regulatory elements


  • Promoter (near the transcription start site)
  • Upstream regulatory regions
• Transcription unit for Eukaryotic genes includes 5' UTR, Exons, Introns, 3'UTR
• In bacteria, multiple ORFs that encode different proteins can be transcribed together
• Overview of the Three Phases of Transcription

  1. Initiation

  2. Elongation

  3. Termination
• Bacterial RNA Polymerase


  • Bacteria has only one type of RNA polymerase, which makes all the RNAs (mRNA, tRNAs, rRNAs)
  • Multi-subunit enzyme. The core of the enzyme is made of 5 subunits: 𝛼2ββ'ω. This core is active in vitro, meaning it can transcribe RNA in a test tube if given a DNA template
  • A σ factor protein is required to initiate transcription in vivo. The σ factor binds to the promoter region and recruits the 5-subunit RNA polymerase core
• Initiation of transcription in bacteria


  1. The σ factor binds to two regions in the promoter located around position -35 and position -10

  2. There are different σ factors and each recognizes a different sequence and initiates the transcription of different classes of genes
• Promoters that more closely match the consensus sequence will be transcribed at a higher rate
• DNAse Foot Printing


  1. Clone the promoter sequence into a plasmid. Linearize the sequence and radiolabel at the 5'end or the 3'end

  2. Incubate the DNA binding protein (σ70) with the radiolabeled promoter sequence

  3. Add DNase I, which will cleave the DNA that is not bound to protein

  4. Remove the proteins from the DNA fragments and separate by size on a gel. Visualize the gel using the radioactivity
• Based on the results shown in Slide 18, the difference between the four samples in the DNA Footprinting experiment was the presence or absence of the DNA binding protein (σ70)
• The Mechanism of Transcription in E. coli


  1. Initiation: A σ factor and RNA polymerase bind to the promoter. RNA polymerase makes many abortive transcripts that are 8-9 nucleotides long

  2. Elongation: RNA polymerase holds on and elongation can proceed. The enzyme is processive, so once it gets going, it continues until the transcript is complete

  3. Termination: There are two mechanisms
• Elongation - Chemical Reaction of RNA synthesis


  1. RNA Polymerase Requires: 1) DNA template, 2) Mg2+, 3) four rNTPs (ATP, GTP, UTP, CTP)

  2. Phosphodiester linkage: The 3'OH group initiates a nucleophilic attack on the alpha-phosphate of the incoming rNTP, with concomitant release of a pyrophosphate (PPi) (i = inorganic)

  3. Importance of the Magnesium ions, which are coordinated by negatively charged Asp residues on RNA poly II: 1) Deprotonation (removal of H) of 3'OH group, allowing it to attack alpha phosphate, 2) Neutralizes negatively charged pyrophosphate, thus stabilizing transition state
• Transcription Termination in Prokaryotes


  1. One mechanism depends on Rho, which is a helicase enzyme

  2. The second mechanism is Rho-independent. This mechanism depends on a sequence that makes a hairpin structure in the RNA
• The default state of transcription in E. coli is to be on at a low level. Proteins (transcription factors) are required to repress transcription
• The default state for transcription in eukaryotes is off because DNA is packaged into chromatin. Most transcription factors activate expression