Lectured

• Trans-acting factors


  Usually protein – interact with DNA

  
• Trans-acting factors


  1. Bind DNA directly, or are recruited to DNA by other factors

  2. Influence transcription (positively or negatively), either by directly recruiting polymerase (or blocking it), or indirectly by recruiting other factors

  
• Cis-acting elements


  Sequences on the DNA that are recognized and bound to by trans-factors

  Can contain a single binding site, or a cluster of sites (e.g. enhancers)

  Can be near the start site (i.e. promoters) or far away (e.g. enhancers)

  
• Cis-acting elements


  • Promoters
  • Enhancers
  • Regulatory binding site
  • Regulatory sites
  • Activator/repressor sites
• Cis regulation


  Regulation that occurs when a region only affects transcription of the DNA strand it is on (i.e. it cannot impact transcription of another DNA strand)

  
• Trans regulation


  Regulation that occurs when a factor can affect transcription on any DNA strand

  
• Transcription factors (repressors and activators) bind to regulatory sites (enhancers, promoters, etc.)
• Architectural regulators bind to architectural regulator-binding sites
• Cofactors (coactivators and corepressors) do not bind DNA directly, but can facilitate interactions that activate or repress transcription
• Insulator-binding proteins bind to insulators
• Effectors are small molecules that can bind to transcriptional regulators and affect their function
• Transcription activators


  Can bind to both close or distant regions from the promoters (i.e. enhancers or regulatory sites) and help to recruit transcription machinery to promoters
• DNA looping

  When a transcription factor bound to an enhancer binds to the promoter, it links the two distant regions and forms a loop of DNA between them
• Architectural regulators


  Bind to DNA at architectural regulator-binding sites and alter the structure of the DNA/chromatin, which indirectly facilitates transcription by making it easier for distant transcriptional activators to reach the promoter

  
• Insulators


  Cis-elements that block activators and repressors from being able to affect transcription from one side of the insulator to the other
• Effectors


  Small molecules that can bind to transcriptional regulators and affect their function

  Can help to repress transcription (inhibitor), or they can help to activate transcription (inducers)

  Sometimes metabolites produced by the gene they regulate, and can act in a positive or negative feedback loop
• The lac operon is a classic example of how gene expression is regulated in bacteria
• lacZ, lacY, and lacA


  Encode proteins that metabolize lactose
• lacI and lacO


  Regulate when lac operon is expressed
• Mutations in lacI or lacO lead to uncontrollable expression of Lac genes
• lacI


  Encodes protein called lac repressor
• lacO


  Binding site for lac repressor (called an Operator)
• Lac repressor binds to the operator sequence to block Lac expression
• Inducer


  A type of effector that binds to the lac repressor, blocking its function and allowing lac expression
• Allolactose


  The inducer of the lac operon, a byproduct of lactose metabolism by the lac operon
• The lac repressor binds to 3 lac repressor binding sites on the lac operon: O1, O2, and O3
• Cyclic AMP (cAMP) is over-produced when glucose is absent, but turned off when glucose is abundant
• cAMP-CRP


  Binds to the Lac promoter to activate transcription
• When glucose is high, there's no free cAMP-CMP, so only very low transcription can occur even if lactose is present
• When glucose is low and lactose is high, then the lac repressor is unbound and high transcription occurs
• Regulatory binding site sequences are often inverted repeats
• Helix-turn-helix motif


  One helix (the recognition helix) fits into the major groove of DNA to recognize specific sequences
• Leucine Zipper


  A motif consisting of several leucines spaced about 7 amino acids apart, used to hold two subunits together, not directly involved in DNA binding
• Zinc Fingers


  Motifs held together by Zinc ions, each with a DNA recognition helix, can be stacked together to grip DNA by the major groove
• Inverted repeats


  • CANNTG
  • CANNTG
• DNA binding proteins are often dimers
• How proteins recognize specific DNA sequences

  1. Example

  2. Helix-turn-helix motif

  3. Recognition helix – One helix (red) fits into major groove

  4. Most DNA-binding motifs recognize the major groove, because it has more features to interact with (hydrogen bond donors/acceptors, etc.) than the minor groove

  
• Leucine Zipper


  • A motif consisting of several leucines spaced about 7 amino acids apart
  • Zipper regions hold two subunits together, not involved in DNA binding, but are linked to a DNA binding region
• Zinc Fingers


  • Zinc finger motifs are held together by Zinc ions
  • Each motif has a DNA recognition helix
  • Each motif is weak, but can be stacked together (beyond dimerizing), with each motif acting like a finger, gripping the DNA by the major groove
  • Zinc ion stabilizes structure, but is not directly involved in DNA recognition
• Some proteins contain all 3 motifs