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Lecture 14

Lecture 14 - Prokaryotic Gene Regulation

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Biology 2581B
Jim Karagiannis

LECTURE 13: PROKARYOTIC GENE REGULATION Key Concepts 1. Induction 2. Positive and Negative Control Mechanisms 3. lac operon model 4. Cis/trans test E. coli Biphasic Growth Curve • Interestingly, B-galacotosidase, the enzyme responsible for lactose metabolism is produced in phase II, but not in phase I • The simple monosaccharide glucose, is preferred over the di-saccharide, lactose as a carbon and as an energy source Lactose Utilization in E. Coli • Two genes are critical for the breakdown of lactose into simpler sugars • The lacY gene encodes lactose permease • The lacZ gene encodes B-galactosidase Lactose utilization in an E. coli cell Lactose passes through the membranes of the cell via an opening formed by the lactose permease protein. Inside the cell, B-galactosidase splits lactose into galactose and glucose. The Dilemma Facing Bacteria • Unicellular cell – so must be capable of extreme adaptation to changing environment • Must not waste energy making products it does not need • So must be capable of switching genes ON as needed or OFF when not needed • So gene regulation (ON/OFF and VOLUME) very important • How does E. coli regulate the production of B-galactosidase? Different Genes Need Different Strategies for Control • Catabolic genes are involved with the breakdown of complex products into simpler ones o For example, ability to utilize lactose. So these genes only needed when the complex compound is present. • Lactose is not a desirable sugar, but E. coli will use it as a carbon source if no other choice is available • Therefore, it would be most advantageous for an E. coli cell to express lacZ and lacY when: o Lactose is presentAND o Glucose is absent • The ‘lac’ genes are normally OFF and are actively turned ON (INDUCED) when grown in the presence of lactose • In the absence of lactose B-gal, and lactose permease is present at extremely low levels. • However, when grown in the presence of lactose, the levels of B- gal and lactose permease increase 1000 fold • (Note: a derivative of lactose, called allolactose, is the inducer in this system) • Interestingly, LacZ and LacY localize to a tightly linked cluster of genes on the bacterial chromosome • The lacZ, lacY, and lacA genes are transcribed in unison as part of transcriptional unit referred to as an operon • What is the advantage of this organization? o All genes involved in the same biological process can be regulated together • P = site from which RNApolymerase initiates transcription (promoter) When lactose is present, allolactose, an inducer derived from the sugar, binds to the repressor. This binding changes the shape of the repressor, making it unable to bind to the operator. With the release of the repressor from the operator, RNApolymerase gains access to the lac operon promoter and initiates transcription of the three lactose-utilization genes into a single polycistronic mRNA. The Use of Mutants in Constructing the lac operon Model • Three-letter abbreviations are used to describe phenotypes in E. coli • Lac+ cells: are able to utilize lactose as a carbon and energy source + o Wild type E. coli cells show a Lac phenotype • Lac- mutants: are unable to utilize lactose o Cells carrying loss of function mutations in the lacZ and lacY genes show a Lac phenotype o While of interest, these mutations did not provide any insight into how the lac operon was regulated • Lac- mutants in E. coli o LacY- mutants cannot import lactose o LacZ- mutants cannot catabolize lactose Models of Induction • Alternative 1 – the inducer positively regulates an activator • Alternative 2 – the inducer acts to relieve inhibition of transcription o i.e. the inducer inhibits a repressor Evidence for a Repressor Protein • Constitutive mutants: cells produced B-gal all the time (i.e. when lactose present or absent) • These mutations defined a new gene at a distant chromosomal locus that was called, lacI (“I” because it appeared to be involved in the “inducibility” of the operon) • Suggested a negative regulatory role for the lacI gene product The PaJaMo Experiment: Further Evidence for a Repressor Protein 1. Grow recipient cell (lacI, lacZ) in the absence of lactose. • Cytoplasm devoid of functional lacI and lacZ gene-products 2. Introduce copies of the lacI and lacZ genes into the recipient (this produces a merodiploid • i.e. cell containing two copies of some genes) 3. Monitor the level of B-galactosidase synthesis The PaJaMo Experiment: The Results • Key result: induction is possible even in the absence of
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