Lecture 17 Until now we have been considering mutations that lead to constitutive synthesis of B-galactosidase. It is also possible to get mutations that are uninducible. For example, a mutation in the promoter (Lacp)is uninducible IPTG +IPTg Interpretation PZ- P is uninducible Z+/F P+Z+ P is recessive *P-Z+/F P+Z P is cis-actin PZ/F P+Z+ *Note that this experiment can also be viewed as a complementation test that shows that acP 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 I designates a"super repressorwhich binds to the operator dNa but won't bind saucer 工PTG 工PTG Interpretation z IS is uninducible Z+/F IZ+ 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
Lecture 17 Until now we have been considering mutations that lead to constitutive synthesis of ß-galactosidase. It is also possible to get mutations that are uninducible. For example, a mutation in the promoter (LacP–) is uninducible. –IPTG +IPTG Interpretation P– Z+ – – P–is uninducible P– Z+/ F’P+Z+ – + P–is recessive *P– Z+/ F’P+Z– – – P–is cis-acting ’P– Z–/ F P+Z+ – + *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. –IPTG +IPTG Interpretation IsZ+ – – Isis uninducible IsZ+/ F’I+Z+ – – Isis 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 maltose glucose glucose (glucose-glucose) intracellular maltose MalE transport proteins (amylomaltose) 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 amount MalE 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 maltose +maltose Interpretation Maltose induces Mal operon MalT Malt is uninducible MalT-/F MalT+ Malt is recessive MalT- Ma@*/F MaIT+ MalA MalT is trans-acting MaIT- Mal@-/F MaIT+ Mala+ 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
maltose maltose glucose + glucose (glucose-glucose) intracellular maltose MalQ transport proteins (amylomaltase) 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. log amount + maltose cell mass MalQ 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 MalTmutations with the following results: maltase activity –maltose +maltose Interpretation Mal+ – + Maltose induces Mal operon MalT– – – MalT–is uninducible MalT–’/ F MalT+ – + MalT–is recessive MalT– MalQ+/ F’MalT+ MalQ– – + MalT is trans-acting MalT– ’ MalQ–/ F MalT+ MalQ+ – + 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 activator. As shown in the diagram below, maltose binds to the Mal activator protein causing a conformational change in Malt allowing it to bind near to the promoter and to stimulate transcription. Note that the genes required for maltose uptake are located in an operon elsewhere on the chromosome but these genes are also regulated by MalT The Mal Operon activator maltodextrin protein amylomaltose RNA polymerase Mat P initiator MaIP MalE romoter induc This model requires a site, called the initiator, which is where the activator binds near site should behave in dominance and cis/trans tests, you will see why in practice itis the promoter to activate transcription. If you think about how mutations in an initiate difficult to distinguish initiator site mutations from promoter mutations It is also possible to isolate super activator"mutants that will bind to the initiator site and activate transcription regardless of whether the inducer maltose is present. Such alleles of the MalT gene are called malTe and their properties are given below -maltose +maltose Interpretation Malte is constitutive MalTc/f MalT+ Maltc is dominant MalTe MalE/FMT+MQr◆ MalTe is trans-acting MalTc MaIQ/ FMaIT* MaIQ.◆ For a multimeric activator it should also be possible to isolate activator-d mutants that will interfere with the binding of wild-type subunits to the initiator site. Actually Malm-d mutants have not been isolated, probably because Malt is a monomer
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 activator. As shown in the diagram below, maltose binds to the MalTactivator protein causing a conformational change in MalT allowing it to bind near to the promoter and to stimulate transcription. Note that the genes required for maltose uptake are located in an operon elsewhere on the chromosome, but these genes are also regulated by MalT. The Mal Operon activator maltodextrin MalT MalP MalQ P promoter initiator protein phosphorylase amylomaltase + inducer RNA polymerase (maltose) This model requires a site, called the initiator, which is where the activator binds near the promoter to activate transcription. If you think about how mutations in an initiator site should behave in dominance and cis/trans tests, you will see why in practice it is difficult to distinguish initiator site mutations from promoter mutations. It is also possible to isolate “super activator” mutants that will bind to the initiator site and activate transcription regardless of whether the inducer maltose is present. Such alleles of the MalT gene are called MalTc and their properties are given below. –maltose +maltose Interpretation MalTc + + MalTcis constitutive MalTc’/ F MalT+ + + MalTcis dominant MalTc MalQ+/ F’ MalT+ MalQ– + + MalTcis trans-acting MalTc ’ MalQ–/ F MalT+ MalQ+ + + For a multimeric activator it should also be possible to isolate activator-d mutants that will interfere with the binding of wild-type subunits to the initiator site. Actually MalT-d mutants have not been isolated, probably because MalT is a monomer
