Prokaryotic Gene Regulation

Gene regulation
1. Cause
  1. Variations in env
    1. light, temp, food source
  2. transcription and translation are energy expensive
  3. Products of most genes only needed in certain cells at certain times
2. What
  1. regulation of synthesis of gene's transcript and protein product
    1. adjust transcription of RNA, OR, or translation of mRNA into protein
    2. stability of mRNA and posttranslational modifications of proteins
3. how
  1. binding of regulatory proteins
    1. regulates rate of transcription
    2. to DNA sites adjacent to protein-coding region
4. Genetic switches
  1. specific DNA sequences bound by specific regulatory proteins under specific conditions
  2. Activator
    1. Binds to activator binding site (ABS) upstream of promoter
    2. helps RNA polymerase bind to promoter
    3. Must be present for transcription
  3. Repressor
    1. Binds to operator downstream of promoter
    2. Acts like a road block to prevent transcription
    3. Must be absent for transcription
  4. 2 sites on a protein
    1. Allosteric Effector binding site
      1. Turn on
        Allosteric effector binds
        protein changes shape at DNA binding region
        Can bind to DNA
        Allosteric effector absent
        Cannot bind to DNA
      2. Turn off
        Allosteric Effecto binds
        protein changes shape at DNA binding region
        Cannot bind to DNA
        Allosteric Effector absent
        Can bind to DNA
    2. DNA binding region
E. coli
1. Normal food source is glucose
2. Can use lactose as food source when lac operon is activated
  1. lac operon
    1. Operon
      1. segment of DNA that encodes a multigenic mRNA with a common promoter and regulatory region
    2. CAP binding site on Promoter
      1. Promoter (P)
        ON/OFF
        Off
        P- mutation
        RNA polymerase cannot bind
        No transcription
        mutation in -35 or -10 regions
        On
        P+
        RNA polymerase can bind
        Transcription
        Operator
        ON/OFF
        OFF
        lacI transcribes repressor (I+)
        Repressor I binds to operator
        No transcription
        trans-acting
        mutation lacI transcribes repressor (IS)
        Repressor binds to operator
        Cannot be removed by lactose
        No transcription
        trans-acting
        Mutation in ABS
        ON
        Mutation Oc
        Repressor can never bind
        Transcription
        Cis-acting
        lacI transcribes repressor
        Repressor I binds to operator
        Lactose binds to I
        repressor DNA binding sites changes, cannot bind to Operator
        Transcription
        lacI mutation (I-)
        Repressor protein is nonfunctional
        Transcription
        trans-acting
        Mutation in DNA binding site
        lacZ
        lacY
        lacA
        Function unknown
        ON/OFF
        OFF
        Mutation Y-
        lacY not transcribed
        Permease not produced
        Lactose cannot enter cell
        Inhibitor never blocked
        Cis-acting
        ON
        Y+
        lacY transcribed
        Permease produced
        Lactose can enter cell
        Inhibitor can be blocked
        Cis-acting
        ON/OFF
        OFF
        Mutation Z-
        lacZ not transcribed
        B-galactosidase not produced
        Lactose not broken down
        Cis-acting
        ON
        Z+
        lacZ transcribed
        B-galactosidase made
        breaks down lactose into glucose and malactose
        Cis-acting
        Cis-acting
      2. Volume controls
        Low expression
        High glucose
        little cAMP
        cAMP doesn't bind to CAP
        CAP doesn't bind to promoter
        Low lac operon experssion
        High expression
        Low glucose
        High cAMP
        cAMP binds to CAP AE site
        CAP binds to Promoter
        Helps RNA poly bind to promoter
        Low expression
        Mutation in CAP
        CAP-
        Mutation in DNA binding site
        Can't bind to Promoter
        CAP*
        Mutation in AE site
        Can't bind to cAMP
      3. trans-acting
    3. Important genes not part of operon
      1. lacI transcribes repressor Inhibitor
      2. CAP transcribes CAP
    4. Experiments
      1. Jacob and Monod
        ASSAY
        test to see if/when lac operon is transcribed
        Easy way to turn on/off lac operon
        Isopropyl-B-D-thiogalactosidase (IPTG)
        artificial lactose
        enters through permease
        binds to repressor
        NOT broken down by B-galactosidase!
        Trigger starts gene expression (Induction)
        Trigger cannot change concentration
        Easy way to tell if lac operon was expressed under conditions of assay
        ONPG
        artificial lactose
        enters through permease
        can be broken down by B-galactosidase
        produces yellow product
        Detect formation of product of B-galactosidase breaking down lactose
        strains of E. coli that have two copies of operon
        create "diploid" bacteria
        F' factors carry lac region to produce merozygotes
        Determine dominance, survival, complimentation

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