Chapter 15 posted (1).pptx

Regulation of gene expression in bacteria Outline • What it means to regulate • Models for gene regulation – Lac operon – inducible system – Trp operon – repressible system What do we mean by regulation? • When the gene is “on” or “off” • How much expression? • Where (what cells) the gene is exp...

Regulation of gene expression in bacteria Outline • What it means to regulate • Models for gene regulation – Lac operon – inducible system – Trp operon – repressible system What do we mean by regulation? • When the gene is “on” or “off” • How much expression? • Where (what cells) the gene is expressed • Inducible: Genes that must be turned on – Default is OFF – Lac operon • Repressible: Genes that must be turned off – Default is ON – Trp operon The lac operon • Structural Genes: (lacZ, lacY, lacA) – LacZ: b-galactosidase, lactose metabolism – LacY: permease, allows lactose entry into cell – LacA: transacetylase, function unclear • Regulatory region – Promoter (P) – Operator (O) • Repressor gene (I) 15-1 LacZ Energy (ATP) 15-2 The lac operon • Repressor: trans-acting factor – Encodes protein that binds to operator – Protein also has a lactose binding site • Promoter/Operator: cis-acting factors – DNA elements directly involved in regulation of transcription 15-5 The lac operon, no lactose (default state) genotype 15-5 The lac operon, turning it on 15-5 How mutations in the lac operon affect transcription • I- = no repressor made or operator binding region is altered • Operon is always ON 15-6 How mutations in the lac operon affect transcription • OC = “constitutive” • Operator sequence altered, repressor cannot bind • Operon is always ON 15-6 How mutations in the lac operon affect transcription • IS = “superrepressed” • Lactose cannot bind repressor, repressor stays bound to operator • Operon is always OFF 15-7 How mutations in the lac operon affect transcription • Understand the genotype: – Oc, Is, cis vs.trans etc • Understand how the operon works: – Go step by step through the process, beginning with the repressor: • Is it made? • Can it bind to the operator? • If present, can lactose bind the repressor? – Ask similar questions for other components • Is lactose present? Merozygote! This element is in trans Oc : constitutive Repressor cannot bind LacZ The preferred sweet! Energy (ATP) 15-2 Catabolite-Activating Protein (CAP) • Adenyl cyclase: converts ATP to cAMP • Glucose inhibits adenyl cyclase • CAP + cAMP = CAP-cAMP binds to promoter, permitting transcription • In presence of glucose, no cAMP made, no CAP binding 15-8 Catabolite-Activating Protein (CAP) • • • • Glucose inhibits adenyl cyclase cAMP levels decrease CAP-cAMP cannot bind efficiently RNA polymerase does not bind efficiently 15-8 Putting it all together • High lactose, low glucose: – Operon is ON , converting lactose into galactose and glucose • Now have high glucose, which turns operon OFF • When glucose is depleted, operon turns ON 15-2 Trp operon • Trp operon encodes genes for the production of tryptophan • Repressible system (lac operon inducible) – Default is ON – If no tryptophan present, operon is on • If tryptophan (trp) is present, trp operon is OFF (repressed) – No need to make tryptophan if already present! Trp operon: components • Similar to lac operon in layout • Key differences: – Repressor needs tryptophan to be functional – Attenuator 15-9 Trp operon: default state • Default state: No tryptophan present • Repressor is made, but cannot bind to operator • Result: transcription of structural genes required for tryptophan synthesis 15-9 Trp operon: Tryptophan present • Tryptophan binds repressor • Repressor can now bind operator • Repressed transcription of structural genes (~70 fold) 15-9 Attenuation • Attenuation: – Can further diminish transcription based on the amount of trp present in the cell – ~10 fold (total of ~700 fold with repressor) • Attenuator: – Located in leader sequence – Contains 2 UGG triplets (encodes tryptophan) – If trp is scarce, cannot translate this region 15-9 Attenuation Attenuation • How it works: – Remember, transcription and translation can occur simultaneously in bacteria! – High trp: • Ribosome can translate attenuator • mRNA forms terminator mRNA hairpin: – terminates transcription just after attenuator sequence – Low trp: • Ribosome stalls at attenuator • mRNA forms alternative hairpin (antiterminator) – allows transcription of structural genes Attenuation Contains UGG UGG Anti-terminator hairpin Terminator hairpin Review • What it means to regulate • Models for gene regulation – Lac operon – inducible system – Trp operon – repressible system