Regulation of Gene Expression How are genes turned on and off One of the most fundamental questions in molecular genetics Genes are only turned on when needed Just like there are simple and complicated electronic circuits so there are simple rules underlying the turning on and off of genes but complex modules of these circuits act together in living cells Bacteria regulate genes in response to external and internal conditions In bacteria many genes that are needed together are clustered together in There are also often transcribed together in a single mRNA molecule a operons on the chromosome polycistronic mRNA Proteins from different are present in cells at a large range of concentrations but can vary over time in response to environmental changes Constitutive genes on ALL the time Repressible genes gene on but turned off often by the end product of a biosynthetic pathway they encode Negative control gene is on until actively turned off Positive regulation gene off until actively turned on a given gene may Lactose Metabolism The Lac Operon is regulated by and inducible system be regulated by a combination of both Bi phasic growth curve Jacques Monod found that if glucose and lactose are present in the growth medium the glucose is used first growth phase I in diagram Then the bacteria adjust to the lack of glucose middle lag phase and then switch to using the lactose growth phase II Diauxie Historically it was found that enzymes involved in catabolism of the sugar lactose were not expressed until lactose was added to the medium a phenomenon referred to as induction Lactose is referred to as an effector inducer molecule Genetically it was shown that an element of the DNA near the catabolic genes is involved in controlling the response to the effector molecule a cis acting element Regulatory molecules that bind to the cis acting element are referred to as trans acting factors Figure 15 02 The catabolic conversion of the disaccharide lactose into its monosaccharide units galactose and glucose The investigation of the lac operon was the study that first revealed the nature of gene structure and regulation Lactose is a disaccharide made of glucose and galactose 1 Beta galactosidase lacZ converts lactose to glucose and galactose or converts lactose to allolactose the direct effector molecule 2 Lactose permease lacY transports lactose into the cell through membrane lactose catabolism 3 Transacetylase lacA helps remove toxic byproducts of Mutants of these enzymes mapped VERY close together Figure 15 3 mRNA half life is very short and lac operon is turned off shortly after all When E coli is grown in the presence of glucose NO lactose degrading the lactose is used up enzymes are present In fact no lactose used until all glucose used up like the Pasteur effect in yeast where fermentation increased after ar is used up diauxie even if lactose is present When glucose is absent and lactose present the amount of lac degrading enzymes increase 1000X in direct proportion to the amount of lactose present in the medium Enzymes are induced in similar amounts coordinate induction Gratuitous inducer analogs of lactose IPTG turn on enzymes but they are NOT the substrate of the enzymes idea that induction does NOT occur at enzymatic level The gratuitous inducer isopropyl thiogalactoside IPTG turns on Model for regulation of the lac genes lac operon like lactose does An operon is a cluster of genes plus the operator region itself and the promoter the expressions of which are regulated by operator repressor protein interactions In the absence of lactose the LACI repressor protein as a tetramer binds to the lacO operator sequence and blocks access of the transcription factors to the operon an example of negative control In the presence of lactose some lactose is converted to allolactose an isomer of lactate which binds to the LACI repressor protein and via an allosteric shift causes it to lose its affinity for the operator and it falls off With no repressor present the polycistronic message is produced and translated The components of the wild type lac operon and the response and described in Figure 15 5a the text Figure 15 5b regulation of the wild type lac operon and the response in the absence of lactose Figure 15 5c lactose Regulation of the wild type lac operon and the response in the presence of Genetic Proof of the Lac operon Mutations affecting the regulation of gene expression They also isolated 2 classes of mutants in which the operon was expressed constitutively i e part of the constitution of the cell One of these mapped to a region next to the lacZ named the operator lacO The second mapped a little further way to a region named the repressor lacI Operator mutations By use of partial diploids merozygotes obtained using F strains Phenotype lacZ constitutive but lacY is inducible implies lacOC mutation is cis dominant to lacO and that the lacO DNA region does not encode a diffusible product Figure 15 6a The response of the lac operon in the absence of lactose when a cell bears the I mutant repressor mutation Figure 15 6b The response of the lac operon in the absence of lactose when a cell bear the OC constitutive operator mutation Table 15 1 Mutations in the protein coding genes By using chemical mutagens they were able to isolate mutants that were missing one of the ORFs for lacZ lacY or lacA These mutations were also used to show the order of the genes in the operon lacI regulatory mutations expressions of both operons is inducible in presence of the inducer implies that the lacI gene is trans dominant to the lacI gene lacI produces a diffusible cellular product lacIs is a super repressor that binds lacO even in the presence of the inducer lactose Figure 15 7 LacI was eventually shown to encode a DNA binding protein The response of the lac operon in the presence of lactose in a cell bearing the IS lacIS is a super represent that binds lacO even in the presence of the inducer mutation lactose LacI was eventually shown to encode a DNA binding protein Isolation of the LAC repressor protein Models of the LAC repressor protein The repressor monomer the arrow points to the inducer binding site The DNA binding region is shown in red The repressor dimer bound to 2 21 base pair segments of operator DNA shown in blue Positive Control of the Lac Operon Catabolite Repression Ho is the lac operon turned off when glucose is present but lactose is also present When only lactose is present a protein called