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Genetics Lecture No. 14.docx

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Western University
Biology 2581B
Jim Karagiannis

Genetics Lecture No. 14: Prokaryotic Gene Regulation th Monday March 4 , 2013 E. Coli Biphasic Growth Curve: -At time 0, both glucose and lactose are added to the E. coli media. During Phase I, the E. coli experience growth until glucose is depleted and during Phase II, the E. coli experience growth until lactose is depleted. The simple monosaccharide glucose is preferred over the disaccharide lactose as a better carbon and energy source since it can be used immediately and doesn’t need to be broken down. Interestingly, β-galacotosidase (the enzyme responsible for lactose metabolism) is produced in phase II, but not in phase I. So how do the E. coli cells know to keep the β-galacotosidase gene turned off when glucose is in the media? Lactose Utilization In E. Coli: -Two genes are critical for the breakdown of lactose into simpler sugars: The lacY gene (which encodes lactose permease) and the lacZ gene (which encodes β-galacotosidase). Lactose permease is the membrane protein that is involved in transporting lactose into the cell, while β- galacotosidase is the enzyme responsible for breaking lactose down. The Dilemma Facing Bacteria: -E. coli, as unicellular cells, so must be capable of extreme adaptation to changing environmental conditions, which means not wasting energy making products they do not need. As such, they need to be capable of switching genes on as needed or off when not needed, making the regulation of genes highly important. By what mechanism does E. coli regulate the production of β-galacotosidase? Different Genes Need Different Strategies For Control: -Catabolic genes are involved with the breakdown of complex products into simpler ones (e.g. the ability to utilize lactose) and these genes are only needed when their respective substrate 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 lacY and lacZ when: Lactose is present & glucose is absent. Regulating The LacY & LacZ Genes: -The ‘lac’ genes are normally off and are actively turned on (or induced) when grown in the presence of lactose. In the absence of lactose, β-galacotosidase and lactose permease are present at extremely low concentrations. However, when grown in the presence of lactose, the levels of β-galacotosidase and lactose permease increase 1000 fold. Note of course that the actual inducer of these lac genes is a derivative of lactose, called allolactose. Interestingly, lacZ and lacY localize to a tightly-linked cluster of genes on the bacterial chromosome. Unlike β- galacotosidase and lactose permease, the product of the lacA gene is not required for the catabolism of lactose, which is why the lacA gene product is rarely discussed. The lac A gene encodes lactose transacetylase (biological purpose of acetylation remains unknown). The lacZ, lacY, and lacA genes are transcribed in unison as part of single transcriptional unit (which gives rise to 3 distinct proteins) referred to as an operon (a unit of DNA composed of specific genes, plus a promoter and/or operator that acts in unison to regulate the response of the structural genes to environmental changes). The advantage of this organization is that all the genes involved in the same biological process can be regulated together. The promoter (P) is the site from which RNA polymerase initiates the operon’s transcription. Designing A Genetic Regulatory Switch: -Certain proteins are used to control the ability of RNA polymerase to transcribe the operon. Proteins can either act as repressors (part of negative regulation) that diminish transcription when bound to cis-acting elements or activators (part of positive regulation) that augment transcription when bound to cis-acting elements. Cis-acting elements (or cis-control elements) are short DNA sequences that constitute the control elements adjacent to genes. Through their binding to transcription factors, cis-acting elements control or modulate transcription initiation at one or more nearby genes. Promoters, enhancers, and locus control regions are three types of cis -control elements. 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 (wild type E. coli cells show a Lac phenotype). Conversely, Lac mutants are unable to utilize lactose. Cells carrying loss of function mutations - in the lacY and lacZ genes show a Lac phenotype. These mutations did not provide any insight into how exactly the lac operon was regulated. LacY mutants cannot import lactose into the cell, while lacZ mutants cannot catabolize lactose. Models Of Induction & Evidence For A Repressor Protein: -Originally, two alternatives were suggested as models of induction of the lac operon. Alternative 1 stated that the inducer positively regulates an activator, while alternative 2 stated that the inducer acts to relieve inhibition of transcription (the inducer inhibits a repressor). Constitutive mutants are cells that produced β-galacotosidase all the time (when lactose was present or absent). It was these mutations that defined a new gene at a di
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