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THE PROBLEM

THE PROBLEM. Prokaryotes must accomplish specialized functions in one unspecialized cell Options Have all gene products functioning at all times ( constitutive expression) Turn on genes only as they are needed ( inducible expression) Are examples of both types of expression.

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THE PROBLEM

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  1. THE PROBLEM • Prokaryotes must accomplish specialized functions in one unspecialized cell • Options • Have all gene products functioning at all times (constitutive expression) • Turn on genes only as they are needed (inducible expression) • Are examples of both types of expression

  2. Control of Gene Function • Control mRNA expression and lifetime • Deviations from consensus promoter sequences • Activator proteins • UP elements • REMEMBER: prokaryotic mRNAs are polycictronic, can have several genes involved in a metabolic pathway expressed together (coordinated expression) • Control translation and degradation of protein product • Half-life of protein • Position of cistron in polycistronic mRNA • Shine-Dalgarno deviations

  3. Regulation (cont’d) • Negative regulation—Protein (repressor) inhibits transcription (Ex. LexA). • Inducer– binds to repressor, alters form, reduces affinity for target, allows expression of gene. • Sometimes, small molecule required for repressor activity. • Positive regulation—Activator proteinincreases transcription rate. Generally bound to a smaller signal molecule.

  4. Regulation of Enzyme Activity • Degradation of enzyme • Feedback inhibition– generally a form of allosteric inhibition • Remember: the cell is web of competing pathways.

  5. The lac Operon • Lactose—A disccharide hydrolyzed to glucose and galactose . • Lactose metabolizing enzymes expresse as a polycistronic message ( lacZ, lacY, lacA). • Is an inducible operon. • Consists of • Regulatory components • Structural components

  6. The Players • Regulatory • Promoter (P) • Operator (O) • LacI • Structural • lacZ • lacY • lacA

  7. In The Absence of Lactose Repressor tetramer binds operator, prevents transcription No reason for expression is repressed

  8. In The Presence of Lactose Conformational change caused by inducer reduces affinity of repressor/inducer for operator

  9. Role of CRP·cAMP • Expression of lac operon • (+) Glucose (-) Lactose= No expression • (+) Glucose (+) Lactose= Low to no expression • (-) Glucose (+) Lactose= High expression • When [glucose] is high, [cAMP] is low and vice versa. • Cyclic AMP Receptor Protein forms a complex with cAMP and binds at a site near the promoter. • Strongly increases expression • Mechanism: causes bending of DNA, allows RNA pol 2 points of caontact

  10. CAP·cAMP Mechanism • CAP-sensitive promoters usually weak • CAP·cAMP Bends DNA, allowing RNA pol to bind at two points, stabilizing interaction • May also interact with C-terminal domain of sigma LEGEND: Purple- CAP·cAMP Red- RNA pol Blue- Sigma

  11. Galactose Operon • Regulates catabolism of galactose • 3 cistrons encoding structural proteins • 2 promoters (P1 and P2) • 2 operators • Repressor (gal R)

  12. Gal Operon Regulation • Effect of cAMP levels • CAP·cAMP regulates transcription from two promoters in opposite ways • CAP·cAMP activates from P1, inhibits from P2 when [cAMP] transcribe from P1, when [cAMP] transcribes form P2. • As long as no repression, level of Gal mRNA constant • Regulation • Repressor- product of gal R • Inhibits from both operators • Galactose acts as inducer • If galactose absent, both promoters inactive

  13. Gal Operon • One unit of the galR dimer binds to each operator • Induces conformational change, prevents transcription Possible structuresNote: dimer responsible for repression

  14. Ara Operon • Dual action regulatory protein- AraC • (-) arabinose • Represses • (+) arabinose • Activates • AraI • AraI1 • AraI2 • Operators • AraO1- regulates AraC • AraO2- regulates AraBAD Two operators AraI In absence of arabinose- AraC dimer causes loop by joining I1 and O2. no transcription With arabinose, shape change causes dimer to sit on I1 and I2, allowing transcription

  15. Ara operon 2 • NOTE: CAP·cAMP binding site. Increases transcription. • Autoregulation of AraC • AraC transcribed from Pc. • Pc regulated by O1. • As level of AraC rises, binds to AraO1 and prevents transcription from Pc. • prevents wasteful accumulation of repressor • Is an example of autoregulation • Are other models

  16. Trp Operon • Encodes enzymes necessary for Trp synthesis • encodes a set of anabolic enzymes rather than catabolic enzymes. • Anabolic enzymes are generally turned off by presence of a product (feedback inhibition) • In addition to repression, system shows attenuation, a finer level of control. • Structure • 5 structural genes3 enzymes • Promoter and operator precede structural genes • In absence of Trp, TrpR protein is inactive

  17. Tryptophan Operon Repression • Negative control of operon: • Low tryptophan • No repression • transcription • Positive control of operon: • High tryptophan • Tryptophan (a corepressor) combines with free repressor dimer (aporepressor dimer)=repressor dimer • transcription blocked

  18. Attenuation: A Finer Level of Control • Trp operon expression also regulated by attenuation, a much finer level of control. • Trp operon features • Repression very weak • transcription could occur even in presence of repressor • Very energy expensive • Attenuation increases expression 10-fold • Result: Trp operon expression spans a 700-fold range (from inactive to fully active)

  19. Attenuation Mechanism • Special sequences prior between promoter and structural gene • Trp leader • Has translation start site • 2 Trp codons in a row (very rare) • Trp attenuator • Has transcription termination sequence • These sequences weaken (attenuate) transcription when trp is abundant • Operates by causing premature termination of transcription • REMEMBER: transcription and translation occur simultaneously in prokaryotes

  20. Attenuation Mechanism 2 • Different hairpin configurations • Configuration 1—Two hairpins, 4 stems • Configuration 2- One hairpin, two stems • Configuration 1 is more stable • Translation begins as soon as Trp leader transcript emerges • If Trp is in short supply • Ribosome will stall over Stem 1

  21. If Trp Abundant • Ribosome translates, hits termination codon, falls off • Allows formation of 2 hairpins • One contains intrinsic terminator • RNA pol falls off

  22. If Trp is Scarce- Overriding Attenuation • Ribosome will stall over Trp codons in Trp leader sequence • Double hairpin can’t form, only single hairpin configuration • Allows RNA pol to transcribe through termination sequences

  23. The Operons

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