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

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

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

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

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

The Players Regulatory Regulatory Promoter (P) Promoter (P) Operator (O) Operator (O) LacI LacI Structural Structural lacZ lacZ lacY lacY lacA 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 Expression of lac operon (+) Glucose (-) Lactose= No expression (+) Glucose (-) Lactose= No expression (+) Glucose (+) Lactose= Low to no expression (+) Glucose (+) Lactose= Low to no expression (-) Glucose (+) Lactose= High expression (-) Glucose (+) Lactose= High expression When [glucose] is high, [cAMP] is low and vice versa. 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. Cyclic AMP Receptor Protein forms a complex with cAMP and binds at a site near the promoter. Strongly increases expression Strongly increases expression Mechanism: causes bending of DNA, allows RNA pol 2 points of caontact Mechanism: causes bending of DNA, allows RNA pol 2 points of caontact

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

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

Gal Operon Regulation Effect of cAMP levels Effect of cAMP levels CAP·cAMP regulates transcription from two promoters in opposite ways 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. 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 As long as no repression, level of Gal mRNA constant Regulation Regulation Repressor- product of gal R Repressor- product of gal R Inhibits from both operators Inhibits from both operators Galactose acts as inducer Galactose acts as inducer  If galactose absent, both promoters inactive  If galactose absent, both promoters inactive

Gal Operon One unit of the galR dimer binds to each operator One unit of the galR dimer binds to each operator Induces conformational change, prevents transcription Induces conformational change, prevents transcription Possible structures Note: dimer responsible for repression

Ara Operon Dual action regulatory protein- AraC Dual action regulatory protein- AraC (-) arabinose (-) arabinose Represses Represses (+) arabinose (+) arabinose Activates Activates AraI AraI AraI 1 AraI 1 AraI 2 AraI 2 Operators Operators AraO 1 - regulates AraC AraO 1 - regulates AraC AraO 2 - regulates AraBAD AraO 2 - regulates AraBAD Two operatorsAraI In absence of arabinose- AraC dimer causes loop by joining I 1 and O 2.  no transcription With arabinose, shape change causes dimer to sit on I 1 and I 2, allowing transcription

Ara operon 2 NOTE: CAP·cAMP binding site. Increases transcription. NOTE: CAP·cAMP binding site. Increases transcription. Autoregulation of AraC Autoregulation of AraC AraC transcribed from P c. AraC transcribed from P c. P c regulated by O 1. P c regulated by O 1. As level of AraC rises, binds to AraO 1 and prevents transcription from P c. As level of AraC rises, binds to AraO 1 and prevents transcription from P c.  prevents wasteful accumulation of repressor  prevents wasteful accumulation of repressor Is an example of autoregulation Is an example of autoregulation Are other models Are other models

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

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

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

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

Attenuation Mechanism 2 Different hairpin configurations Different hairpin configurations Configuration 1—Two hairpins, 4 stems Configuration 1—Two hairpins, 4 stems1 Configuration 2- One hairpin, two stems Configuration 2- One hairpin, two stems2 Configuration 1 is more stable Configuration 1 is more stable Translation begins as soon as Trp leader transcript emerges Translation begins as soon as Trp leader transcript emerges If Trp is in short supply If Trp is in short supply Ribosome will stall over Stem 1 Ribosome will stall over Stem 1

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

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

The Operons OPERONINDUCER Repressor gene CAPCOMMENTS laclactoselacI+ One promoter, one operator galgalactosegalR+ Two operators, 2 promoters araarabinosearaC+ araC protein acts as repressor and activator Trp Low Trp trpR (aporepressor) - Anabolic, Trp is corepressor, attenuation