Transcriptional regulationLactose --> glucose + galactoseComponents of the lactose operonMutants in regulatory elements and genes: Interfere with normal regulation of the genes, not the genes themselvesLevels of activity of lactose metabolic enzymesBiology 212 General Genetics Spring 2007Lecture 19: Gene RegulationReading: Chap. 9 pp. 318-326Lecture Outline:1. Transcriptional control 2. Lactose operon: negative control3. Lactose mutants4. Positive control (optional)Lecture:1. Transcriptional controlGene regulation: Control of when, where and under what conditions genes are transcribed and translated.Transcriptional regulation- Transcribe gene when product is needed, turn off transcription when product is not needed.- Efficient way of controlling gene expression—don’t have to commit to processes of transcription and translation.Operon: Group of genes that are coordinately regulated in bacteria. Usually, a single polycistronic mRNA is produced from which multiple proteins are translated. 2. Lactose operon- E. coli prefers to use glucose as a carbon source.- Can use lactose as a carbon source, if necessary.- Need to induce (switch on) gene expression.Lactose metabolismLactose is a disaccharide (made of two sugar subunits)Lactose --> glucose + galactoseNeeded for lactose metabolism:- β-galactosidase: Enzyme that breaks down lactose into glucose and galactose- Permease: Protein that transports lactose into cell- Transacetylase: Enzyme, exact function unknown1Components of the lactose operonStructural genes: Genes that code for proteins- Lac Z: gene for β-galactosidase- Lac Y: gene for permease- Lac A: gene for transacetylaseRegulatory genes and elements: Control expression of the structural genes- Lac I: gene for repressor protein; shuts off the operon- Lac O: operator=element on DNA that repressor binds- Lac P: promoter=site where transcription beginsInducer: Chemical signalling molecule- Form of lactose (allolactose)Organization of the lac operon Fig. 9.4 I P O Z Y AWhen lactose is absent, glucose is present --> Operon is OFF1) I gene is transcribed and translated to give repressor protein2) Repressor binds to operator3) Transcription is prevented4) Operon is off (repressed)When lactose (inducer) is present, glucose is low or absent --> Operon is ON1) I gene is transcribed and translated to produce repressor2) Repressor binds inducer (form of lactose)3) Conformation of repressor is altered4) Repressor can’t bind operator5) RNA polymerase can bind promoter and transcribe genes in the operon (ZYA)6) The polycistronic mRNA is translated to give β-galactosidase, permease and transacetylase proteins.7) Lactose is transported into the cell and utilized as a carbon source.3. Lactose mutants- E. coli strains with mutations in the lactose metabolic genes were used by Jacob and Monod in the early 1960’s to elucidate the lac operon.2- Partial diploid strains, produced when an F factor integrates into the chromosome (form Hfr), but is excised imprecisely, taking some bacterial genes with it. Mutants in structural genes:Lac Z-Lac A- No protein or defective protein producedLac Y-Mutants in regulatory elements and genes: Interfere with normal regulation of the genes, not the genes themselvesLac I- = lac Ic No repressor protein, constituitive expression of lactose operon genesOperon is always ON; no inducer requiredLac OcOperator constituitive mutant; can’t bind repressorOperon is always ON; no inducer requiredLac P- Promoter mutant; no transcription of lactose operon genesOperon always OFFHow do these mutations affect lactose operon expression singly and in combination?Table from Problem 9.16Levels of activity of lactose metabolic enzymesGenotype Uninduced Uninduced Induced InducedZ Y Z YI+O+Z+Y+ 0.1 0.1 100 100I+OcZ+Y+ 25 25 100 100Genotypes of partial diploidsProblem 9.16 part (b)F’ I+P+O+Z-Y-/I-O+Z+Y+What do these symbols mean?F’ = F factor containing some bacterial genesSecond genotype =bacterial chromosomeHow is F’ formed?F factor integrates into bacterial chromosome (forms Hfr); when it comes out, sometimes it takes some of the adjacent bacterial genes with it, creating an F’.3What would be the phenotype of this cell?- I+ is produced in cell from F’.- I+ binds to operator on the chromosome (repressor is diffusable and can act in trans)- In the absence of inducer, the operon is OFF.- When inducer (lactose) is added, repressor binds to the inducer and is unable to bind the operator. The operon is ON.Phenotype is wild type.4. Positive control (may not get to in lecture)To be fully on, the lactose operon also needs a positive regulator. - The positive regulator is highly sensitive to glucose levels in the environment.- Glucose has the effect of repressing the lac operon even when lactose is present.- If glucose is present --> cAMP concentration is lowCRP = cyclic AMP receptor protein: binds to cAMP- Complex binds to the lac operon at a site near the promoter (see Fig. 9.7)- Enhances binding of RNA polymerase to the promoter.High glucose low cAMP low transcriptionLow glucose high cAMP more