Topic 4 - #14 The Lactose Operon

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1 Topic 4 - #14 The Lactose Operon The Lactose Operon The lactose operon is an operon which is responsible for the transport and metabolism of the sugar lactose in E. coli. - Lactose is one of many organic molecules E. coli can use as a carbon and energy source. - However, glucose is the preferred carbon source for E. coli - If we supply E. coli with both glucose and lactose, the cells use the glucose until it is exhausted, stop growing briefly, then start growing again using the lactose. - E. coli cells are grown on a medium containing both glucose and lactose, and the bacterial density (number of cells/ml) is measured. DIAUXIC growth is observed (cellular growth in 2 phases) - During the no growth period, the cells have been adjusting to the new nutrient source by turning on the lac operon and accumulating the enzymes needed to break down the lactose. Enzymes needed for lactose metabolism in E. coli: - Lactose permease transports lactose into the cell (in the outer membrane of the cell) - Beta-galactosidase breaks lactose (disaccharide) down to its component sugars: glucose and galactose (monosaccharides) (Degradative/catabolic reaction)

2 The is lactose operon controlled by on-off regulation: How we would determine or illustrate that regulation: - is growing the bacteria, E. coli, in a nice medium in which sugars are present (lactose environment) and then add to into a flask that divides the two: - Without lactose = very low concentration of beta-galactosidase and lactose permease proteins in the cells - With lactose = very high concentration of beta-galactosidase and lactose permease proteins in the cells This is an INDUCIBLE system. The lactose (derivative allolactose) is an INDUCER of the production of the two enzymes. An inducer is a small molecule that stimulates the synthesis of an inducible protein. The reactions of beta-galactosidase:

3 - The main reaction catalysed by beta-galactosidase is the hydrolysis of lactose (1,4 linkage) - It also catalyses a minor reaction that converts lactose to allolactose (1,6 linkage) - Allolactose acts as the inducer of beta-galactosidase synthesis The Lactose Operon Diagram - Contains a double-stranded DNA genome part of a circular bacterial chromosome - Consists of structural genes genes that transcribe and translate into structural proteins which make up parts of cells in structure etc. - Consists of regulatory elements are upstream, contains the promoter and the operator - Structural genes are transcribed to a single stranded polycistronic mrna. The Z gene translated to betagalactosidase enzyme, while Y gene translated to lactose permease. The A gene translates to transacetylase A. - The I gene at the far left next to regulatory elements, is a gene that is transcribed by its own promoter and transcribed to a single stranded mrna (not polycistronic), which is translated to produce a repressor protein (repressor I). This part of the double-stranded genome is not part of the operon itself. Summary of diagram - In the lac operon, the main operator is adjacent to the promoter - The function of the laca gene product (transacetylase A) is not wellunderstood appears not to be required for lactose catabolism - The laci gene is upstream (5 ) of the operon. It is transcribed from its own promoter and translated separately, to give the repressor protein.

4 Negative regulation of the Lac Operon - When there is NO lactose in the surroundings, the enzymes are not needed and are switched OFF - When the inducer lactose IS present, the enzymes are needed and switched ON - Allolactose acts as an INDUCER - Inducer binds to repressor protein, alters repressor conformation, prevents repressor binding to operator site on DNA How does the Lac Repressor prevent transcription of the Lac Operon? - The lac repressor is a tetramer (has 4 parts) with 2 identical binding sites - The lac operator actually has 3 sites: O 1, O 2, O 3. The O 1 is the main operator site - The repressor binds O 1 and either O 2 or O 3, forming a DNA LOOP - The loop contains the -35 and -10 binding sites recognized by RNA POLYMERASE - These sites are now inaccessible to RNA polymerase - The repressor and the operator binding is not permanent. The right vicinity is determined by the dynamic system at which if there is detach of repressor to operator, due to O 1 strong binding, the reaction to attach back is quick and flexible to join to either O 2 or O 3 How does this compare to: Negative regulation of the Tryptophan operon

5 - When there is NO tryptophan present on the surroundings, the genes are switched ON - When there IS tryptophan present and it enters the bacterial cell, the enzymes are no longer needed and are switched OFF - Difference is because the lac operon is catabolic (degradative) operon, while the trp operon is an anabolic (biosynthetic) operon. Summary: Negative Regulation of the Lactose Operon - Lactose metabolism in E. coli is carried out by 2 proteins, betagalactosidase and lactose permease - The genes for these and transacetylase are clustered together and transcribed from one promoter, giving a polycistronic mrna, i.e. they form an operon - Negative control of the lac operon works by a repressor protein binding to the operator and preventing RNA polymerase from binding to the promoter: no transcription - When lactose is present, its derivative allolactose acts as an inducer by binding to the repressor causing it to dissociate from the operator: transcription of the structure genes occurs End of Topic 4 - #14 The Lactose Operon