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 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 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. Regulation of Enzyme Activity Degradation of enzyme Feedback inhibition– generally a form of allosteric inhibition Remember: the cell is web of competing pathways. 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 The Players Regulatory Promoter (P) Operator (O) LacI Structural lacZ lacY lacA In The Absence of Lactose Repressor tetramer binds operator, prevents transcription No reason for expression is repressed In The Presence of Lactose Conformational change caused by inducer reduces affinity of repressor/inducer for operator 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 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 Galactose Operon Regulates catabolism of galactose 3 cistrons encoding structural proteins 2 promoters (P1 and P2) 2 operators Repressor (gal R) 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 Gal Operon One unit of the galR dimer binds to each operator Induces conformational change, prevents transcription Possible structures Note: dimer responsible for repression Ara Operon Dual action regulatory protein- AraC (-) arabinose Represses (+) arabinose Activates AraI AraI1 AraI2 Two operators AraI In absence of arabinose- AraC dimer causes loop by joining I1 and O2. no transcription Operators AraO1- regulates AraC AraO2- regulates AraBAD With arabinose, shape change causes dimer to sit on I1 and I2, allowing transcription 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 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 genes3 enzymes Promoter and operator precede structural genes In absence of Trp, TrpR protein is inactive 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 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 700fold range (from inactive to fully active) Attenuation Mechanism Special sequences prior between promoter and structural gene Trp leader Trp attenuator Has translation start site 2 Trp codons in a row (very rare) 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 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 If Trp Abundant Ribosome translates, hits termination codon, falls off Allows formation of 2 hairpins One contains intrinsic terminator RNA pol falls off 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 The Operons OPERON INDUCER Repressor CAP COMMENTS gene One promoter, one operator lac lactose lacI + gal galactose galR + Two operators, 2 promoters ara arabinose araC + araC protein acts as repressor and activator Trp Low Trp - trpR (aporepressor) Anabolic, Trp is corepressor, attenuation