Bio 99 Final Lec 13

  1. 7 stages where gene regulation can occur
    • 1.) Transcription Initiation
    • 2.) Posttranscriptional Processing
    • 3.) RNA Stability
    • 4.) Translation 
    • 5.) Protein Modification
    • 6.) Protein Transport
    • 7.) Protein Degradation
  2. 2 type of "things" that regulate transcription
    • trans-acting factor (usually protein)
    • cis-acting elements (DNA)
  3. Trans-acting Factors (usually protein)
    • transcription factors (or sigma factors)
    • transcription activators/repressors
    • transcriptional regulators
  4. Trans-acting factors do 2 things:
    • 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
  5. Cis-acting elements (DNA) (5)
    • promoters
    • enhancers
    • regulatory binding site
    • regulatory sites
    • activator/repressor sites
  6. Sequences on the DNA that are recognized and bound to by trans-factors... (2)
    • 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)
  7. Transcriptional regulation can occur in __ or ___
    cis or trans
  8. You create a mutant cell line that is unable to express Gene X.
    How do you determine whether the mutation is in the promoter (or other
    cis-acting element) for Gene X, or in a trans-acting factor that positively
    regulates transcription of Gene X?
    Introduce a 2nd copy of Gene X and see whether its expression is affected by the mutation
  9. Mutation in the Cis-acting element
    • Regulation in cis occurs when a region only affects transcription of the DNA strand it is on (i.e. it cannot impact transcription of another DNA strand). Regulatory sites on the DNA act in cis.
    • Image Upload 1
  10. Mutation in the Trans-acting factor
    • Regulation in trans occurs when a factor can affect transcription on any DNA strand. Transcription factors act in trans.
    • Image Upload 2
  11. You create a mutant cell line with no expression of Gene X. You introduce a second copy of Gene X in a plasmid (complete with Gene X’s promoter). The second copy of Gene X is expressed. Is the mutation in
    A.) a cis-acting element
    B.) a trans-acting factor
  12. You run a Northern blot and probe with Gene X (will detect both
    copies). If the mutation was in a trans-acting factor, which one would your blot look like?
    Image Upload 3
  13. Trans-acting factors bind to..... (5)
    • transcription factors (repressors and activators)
    • architectural regulators
    • cofactors (coactivators and corepressors)
    • insulator-binding proteins
    • effectors
  14. cis-acting elements bind to.... (3)
    • regularoty sites (enhancers, promoters, etc.)
    • Architectural regulator-binding sites
    • insulators
  15. Transctiption factors: repressors...
    negatively regulate transcription by competing with RNA pol or other activators for binding to the promoter
  16. Transcription Factors: Activators ....
    positively regulate transcription by recruiting or enhancing recruitment of the transcription machinery to the  promoter
  17. Describe how transcription factors can act from long distances (eukaryotic)
    • Transcription activators bind to distant regions from the promoters (i.e. enhancers or regulatory sites) and help to recruit transcription machinery to promoters. Enhancers can be very, very far away from the promoter, like megabases
    • causes DNA looping
  18. DNA looping (eukaryotic)
    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
  19. Architectural Regulators (eukaryotic)
    Architectural regulators bind to DNA at architectural regulatorbinding 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.
  20. Cofactors
    Cofactors (coactivators and corepressors) are proteins that do not bind DNA directly, but can facilitate interactions that activate or repress transcription
  21. Insulators (eukaryotic)
    • Insulators are cis elements that block activators and repressors from being able to affect transcription from one side of the insulator to the other.
    • Also called boundary elements.
    • How they do this is still unclear. They probably block DNA looping.
  22. Effectors
    • Effectors are small molecules that can bind to transcriptional regulators and affect their function.
    • • Effectors (as a class) can help to repress transcription (inhibitor), or they can help to activate transcription (inducers)
    • • Effectors are sometimes metabolites produced by the gene they regulate, and can act in a positive or negative feedback loop
  23. Which trans-acting factor both binds to a cis-acting element as well as directly recruits RNA polymerase?

    D. )
  24. Proteins that act by bridging activators and RNA polymerase are called:

    D. )
  25. The lac Operon (image)
    Image Upload 4
  26. lacZ, lacY, and lacA –
    encode proteins that metabolize lactose
  27. lacI and lacO -
    regulate when lac operon is expressed
  28. Mutations in lacI or lacO lead to uncontrollable expression of....
    Lac genes
  29. lacI and lacO are important for ______ lac operon expression
  30. lacI
    encodes protein called lac repressor
  31. lacO
    binding site for lac repressor (called an Operator)
  32. Lac repressor binds to the operator sequence to block....
    Lac Expression
  33. Does lacO act in cis or in trans?
    A. Cis
    B. Trans
  34. Does lacI act in cis or in trans?
    A. Cis
    B. Trans
  35. In the absence of lactose, .....
    the lac repressor blocks transcription
  36. In the presence of lactose, .....
    the lac repressor is blocked, allowing transcription
  37. An inducer (a type of effector)
    binds to the lac repressor, .....
    • blocking its function and allowing lac expression
    • It’s called an inducer because it induces the operon, not because it induces the repressor (i.e. the inhibitor of an inhibitor is an activator)
    • The inducer is called allolactose
  38. Allolactose
    • allolactose is a byproduct of lactose metabolism by the lac operon
    • when allolactose is produced, it blocks the lac repressor allowing lac expression
  39. What a minute?! You need the lac genes
    to be turned on in order to produce
    allolactose, but allolactose is required to
    induce the lac genes. So how do you turn
    on the lac genes if your inducer requires
    them to already be on?!!!
    The lac repressor isn’t 100% efficient, and  a low basal level of the lac genes are always expressed, producing just enough allolactose when lactose is present to block the lac repressor.
  40. There are actually 3 lac
    repressor binding sites on the lac operon:
    O1, O2, and O3
  41. O_  is the original and is the only one required
  42. The lac repressor binds to O1 and either O2 or O3, creating
    a loop
  43. The loop physically blocks ___ _______
    RNA polymerase
  44. What would happen if you mutated lacZ?
    A. You wouldn’t be able to metabolize lactose, but the
    other Lac genes would still be expressed
    B. The lac operon would become constitutively expressed
    C. You couldn’t make allolactose, and couldn’t stop the lac
    D. The same effect as if you mutated lacI or lacO
  45. Lactose is not as efficient an energy source as ....
  46. How does the lac operon know when glucose is present?
    Cyclic AMP (cAMP) is over-produced when glucose is absent, but turned off when glucose is abundant
  47. When glucose is not there, cAMP ....
    is how the cell tells Lac operon and other sugar metabolizing operons that it’s time to turn on. cAMP is an inducer of Lac expression.
  48. cAMP binds to a protein called _______ to
    activate it
    • cAMP responsive protein (CRP)
    • CRP-cAMP binds to the Lac promoter to activate transcription (i.e. it’s an activator)
    • In the absence of lactose, the lac repressor is still bound, preventing CRP cAMP from turning on the lac genes when there’s  nothing for them to metabolize.
  49. glucose high, cAMP low, lactose _____
    • absent
    • In the presence of glucose, there’s no cAMP-CRP, so no effect on the lac operon.
    • no gene expression
  50. glucose low, cAMP high, lactose ____
    • absent
    • In the absence of glucose, cAMP-CRP can bind to the Lac promoter, but if lactose is also absent, the lac repressor blocks cAMP-CRP
    • no gene expression
  51. glucose high, cAMP low, lactose ____
    • present
    • When glucose is high, there’s no free cAMP-CMP. If lactose is also present, the lac repressor is blocked, but the positive signal given by cAMP-CMP is not there to help and only very low transcription can occur.
    • low level of gene expression
  52. glucose low, cAMP high, lactose ______
    • present
    • When glucose is low, cAMP-CMP bind to the lac promoter. If lactose is high, then the lac repressor is unbound and high transcription
    • high level of gene expression
  53. X-gal
    • a substrate for lacZ (b-galactosidase)
    • • Turns blue when lacZ is active
    • • Used to measure lacZ expression
  54. IPTG
    an inducer, binds to lac repressor to block it, allowing lac expression
  55. researchers wanted to study mutations in the lac operon. They treated their mutants with IPTG to activate the operon, then added X-gal to visualize lacZ expression. In their mutant, the cells did not turn blue, indicating a problem with ....
    lac expression
  56. Which of the following statements about regulation of the lac operon is true?
    A. When glucose is present, gene expression is activated by lactose.
    B. The amount of glucose in the growth medium does not affect the
    level of gene expression activated by lactose.
    C. The binding of the Lac repressor to the operator is not affected by
    the presence of glucose.
    D. Gene expression is high when the Lac repressor is not bound to the
    operator and glucose is available.
  57. You are studying the lac operon, and want to switch on expression
    of the lac operon, by adding something to your media. Which of the following would not work to turn on the lac operon? (assume there’s no glucose in your media)

    D. )
  58. How do proteins recognize specific DNA sequences?
    Regulatory binding site sequences are often inverted repeats
  59. Why?
    because DNA binding proteins are often dimers
  60. Helix-turn-helix motif: Recognition helix
    one helix, fits into major groove
  61. Most DNA-binding motifs
    recognize the ____ groove, because it has more features to interact with (hydrogen bond donors/acceptors, etc.) than the minor groove
  62. Another helix-turn-helix motif: Homeodomain
    • Dimer of helix-turn-helix protein
    • Each dimer contains one helix  that binds to DNA
  63. 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
    • not part of DNA recognition but involved in dimerization
  64. 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
  65. Transactivation domains
    • Transactivation domains are diverse and don’t share common motifs
    • These domains are on separate regions of the protein, allowing researchers to separate them (and swap them). This demonstrates the modular nature of regulatory proteins.
  66. Transactivation domains have 2 basic domains:
    • 1) DNA binding domain
    • 2) Transcription-activation domain
  67. Which sequence is an inverted repeat?

    C. )
  68. What region of each of the 3 major motifs actually recognizes the DNA?

    E. )
  69. You synthesize a fusion protein with JEL4 and RexA. You create a reporter construct to express lacZ, but containing the promoter for JEL4 and the binding site for RexA. Which domains should you use for JEL4 and RexA fusion protein?
    A. Transactivation domain for JEL4, DNA-binding domain for RexA
    B. Transactivation domain for RexA, DNA-binding domain for JEL438
Card Set
Bio 99 Final Lec 13