mid 24 - Control of Gene Expression in Prokaryotes Page...

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Chapter 4 Control of Gene Expression in Prokaryotes Page 115-119
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The Lac Operon • Bacteria also regulate the expression of their proteins. -Example: Synthesizing enzymes required to break down different carbon sources for energy (sugar metabolism). maltose rhamnose galactose lactose glucose In an environment containing both glucose and lactose, E.coli cells preferentially metabolize glucose to make ATP.
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1) The Lac Repressor: Negative Control • The lac repressor (LacI) is a molecule with two recognition sites: 1) One site that recognizes the specific operator sequence for the lac operon 2) Another distinct allosteric site that can bind lactose and certain analogs of lactose ( IPTG ). LacI Allosteric site Z Y A
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2) Catabolite Control or Positive Control • Therefore, if both glucose and lactose are present, synthesis of β - galactosidase is not induced until all of the glucose has been utilized. Why does the cell do this? • This second system of control exists because cells favor the uptake and metabolism of glucose over lactose. > glucose lactose
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Catabolite Control or Positive Control • Once glucose is depleted from the media, E.coli cells respond by synthesizing c yclic a denosine m onop hosphate (cAMP) cAMP Therefore…as [glucose] decreases [cAMP] increase
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Catabolite Control or Positive Control • As the concentration of cAMP increases, it binds to a site in each subunit of a dimeric c atabolite a ctivator p rotein (CAP) protein, causing a conformational change that allows the protein to bind to the CAP site in the lac transcriptional control region. • The DNA-bound cAMP/CAP complex interacts physically with RNA polymerase and increases the affinity of RNA polymerase for the lac promoter. Lac promoter lac Z lac Y lac A Lac I O CAP + lactose - Glucose (high cAMP)
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Lac promoter lac Z lac Y lac A Lac I O CAP + lactose + glucose (low cAMP) Lactose The Lac operon: Example of an inducible operon • + lactose, + glucose: lactose binds to lac repressor and induces conformational change that inactivates the repressor • Inactive lac repressor Pol- σ 70 can proceed to transcribe • Glucose metabolism is favored over lactose for ATP production. • High glucose lowers cAMP accumulation, resulting in low or no CAP activity and low transcription transcription of lac operon.
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Polycistronic mRNA Summary
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Nucleotide sequence of binding sites in the control region of the lac operon cAMP-CAP binding site • cAMP-CAP complex recognizes and binds to a specific site in the control region • Binding of CAP causes conformational change, allowing RNA polymerase to transcribe operon
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The Lac operon: It all comes together • High lactose, lack of glucose, get transcription of lac operon… β -galactosidase [glucose] [galactose] β -gal cleaves lactose into galactose & glucose (catabolites) • Lactose levels decreases and it dissociates from the lac repressor becomes active and binds to operator As glucose levels increases, cAMP levels decreases and dissociates from CAP inactivating it.
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Biochemical Assay to assess
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