Genetics Lecture Notes 7 03 2005 Lectures 17 19 Lecture 17 Gene Regulation We are now going to look at ways that genetics can be used to study gene regulation The issue is how cells adjust the expression of genes in response to different environmental conditions The principles of gene regulation were first worked out by Jacob and Monod studying the E coli genes required for cells to use the sugar lactose as a nutrient lactose glucose galactose LacY permease lactose intracellular glucose galactose LacZ b galactosidase cell mass log amount LacY or LacZ lactose time The logic of the Lac operon is that the proteins required to use lactose are only made when their substrate lactose is available This prevents wasteful expression of enzymes when their substrates are not available At first scientists noted that lactose is both an inducer and substrate for the enzymes of the Lac operon and they therefore concluded that lactose was somehow acting as a template for the formation of the enzyme Then compounds were discovered that could act as inducers but were not themselves substrates for the Lac enzymes The classic example of such a gratuitous inducer is IPTG which is an effective inducer of LacZ expression but isn t hydrolyzed by galactosidase CH3 IPTG galactose S CH CH3 The existence of compounds such as IPTG shows that recognition of the inducer is a separate molecular event from lactose breakdown The next major finding was the discovery of LacI mutants LacI mutants are constitutive meaning that they always express galactosidase at high levels regardless of whether there is an inducer present or not LacI mutants have apparently lost a component of the machinery the cell uses to turn off galactosidase expression The regulatory system turns out to be quite simple and by isolation of mutants and simple genetic tests Jacob and Monod were able to figure out the following scheme The Lac Operon repressor protein b galactosidase permease RNA polymerase P O promoter operator LacI LacZ LacY LacA inducer The idea is that the inducer has a net positive effect on expression because the inducer is a negative regulator of the repressor which is itself a negative regulator of the gene for galactosidase We will now consider how regulatory mutants can be analyzed genetically We will use as examples different mutations in the Lac system but the genetic tests are very general and can be applied to most regulatory systems Dominance test I Z I Z I Z F I Z I Z I Z F I Z I Z F I Z galactosidase IPTG IPTG Interpretation I is constitutive I is recessive Z is uninducible Z is recessive I and Z mutations complement the mutations are in different genes A second type of constitutive mutant inactivates the operator site and is known as a LacOc mutation LacOc mutations are dominant as revealed in tests of the appropriate merodiploids O c Z Oc Z F O Z IPTG IPTG Interpretation Oc is constitutive Oc is dominant You might think that on the basis of a dominance test we could tell whether we have a LacOc or a LacI mutation However life is not so simple because it is possible to find LacI mutations that are dominant Such mutations are known as LacI d They are dominant because the repressor protein is a tetramer and LacI d mutant subunits can combine with normal subunits and interfere with their function I d Z I d Z F I Z IPTG IPTG Interpretation I d is constitutive I d is dominant We will now consider a new genetic test that will let us distinguish LacOc operator constitutive from LacI d dominant repressor negative mutations Cis trans test I O Z IPTG IPTG I d Z F I Z cis I d Z F I Z trans Oc Z F O Z cis Oc Z F O Z trans Interpretation I d is dominant in cis or in trans with Z Therefore we say it is trans acting Oc is dominant only in cis with Z Therefore we say it is cis acting If a mutation is cis acting we take this as evidence that the mutation affects a site on DNA like an operator If a mutation is trans acting we take this as evidence that the mutation affects a diffusible gene product such as a repressor Lecture 18 Until now we have been considering mutations that lead to constitutive synthesis of galactosidase It is also possible to get mutations that are uninducible For example a mutation in the promoter LacP is uninducible P Z P Z F P Z IPTG IPTG P is recessive P is cis acting P Z F P Z P Z F P Z Interpretation P is uninducible Note that this experiment can also be viewed as a complementation test that shows that LacP and LacZ are mutations in the same gene This fits with our primary definition of a gene as the DNA segment needed to make a protein since the promoter is certainly needed for protein expression Promoter mutants in Lac operon can be distinguished from simple LacZ mutations since promoter mutations affect the LacY and LacA genes as well Is designates a super repressor which binds to the operator DNA but won t bind inducer I s Z Is Z F I Z IPTG IPTG Interpretation Is is uninducible Is is dominant Positive regulation Now we will consider how a different E coli operon is regulated The Mal operon encodes several genes necessary to take up and degrade maltose a disaccharide composed of two glucose residues maltose glucose glucose maltose intracellular maltose transport proteins MalQ amylomaltase glucose glucose Much like the Lac operon the products of the Mal operon are induced when maltose is added to cells Thus maltose acts as an inducer cell mass log amount MalQ maltose time When mutants that affect the regulation of the Mal operon were isolated the most common type consisted of uninducible mutations in a gene known as MalT We can apply dominance tests and cis trans tests to MalT mutations with the following results maltase activity Mal MalT MalT F MalT MalT MalQ F MalT MalQ MalT MalQ F MalT MalQ maltose maltose Interpretation Maltose induces Mal operon MalT is uninducible MalT is recessive MalT is trans acting From this table it looks as if the MalT trait is not expressed either in cis or in trans Because MalT is recessive it makes more sense to consider the properties of the dominant MalT allele in the cis trans test Viewed in this way the MalT trait is expressed in both cis and trans and therefore MalT is considered to be trans acting This behavior is different from any of the Lac mutations that we have discussed The interpretation is that MalT encodes a diffusible gene product not a site on DNA that is required for activation of transcription of the Mal operon This type of gene is usually called an
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