Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Bacterial gene regulation: the lac operonLecture outline1. Why does E. coli modify lac operon gene expression in response to its chemical environment?2. Structure and function of the lac promoter3. Molecular mechanisms regulating lac operon transcription✦Transcription factor proteins: LacI and CAP✦Interaction of transcription factors with RNAP✦Regulation of transcription factors by environmental signals (allostery)✦Integration of positive and negative regulatorsIn the early 1960s François Jacob (left) and Jacques Monod (center) discovered the lac operon and developed the first molecular model for how gene expression can be regulated by a cell’s environment. In 1965, they were awarded the Nobel Prize with Andre Lwoff.Escherichia coli lives inside the lower intestine of mammals, where it is immersed in a rich medium of diverse nutrients.E. coli metabolism•E. coli can metabolize a wide variety of organic compounds to obtain carbon and energy. •Growth is the most rapid and energetically efficient using glucose as the primary nutrient.•E. coli can modify its gene expression to optimize energy efficiency:✦When glucose is abundant, the cell represses expression of enzymes used to metabolize other nutrients. ✦When glucose is absent, the cell expresses enzymes needed to metabolize whatever other nutrients are available.Lactose - - + +Glucose + - + -The enzymes responsible for lactose metabolism show this pattern of regulation:•Expression is increased when lactose is available.•Expression is decreased when glucose is also available.When it is metabolizing the disaccharide lactose, E. coli synthesizes the following three proteins:•Lactose permease - membrane transport protein that allows lactose to enter the cell. •b-galactosidase - enzyme that cleaves disaccharide lactose into glucose and galactose for further metabolism. •Thiogalactoside transacetylase - enzyme that inactivates certain toxic sugars that can enter the cell through lactose permease.β-GalactosidaseLactose permeaseThiogalactosidetransacetylaseThe lac operon5’+1lacZ lacY lacAPromDNARNA3’AUG AUG AUGstop stop stopProteinsNOTE: a molecular biology convention is that specific genes and RNAs are usually named in italics; proteins are named with roman (= non-italic) letters.Expression of the lac operon is primarily regulated at the level of transcription.If lactose is added to the growth medium of starved cells, it induces a 1,000-fold increase in B-galactosidase expression. There is a similar increase in the other two lac operon gene products as well.+1lacOTranscriptionTranslationlacIlacZ lacY lacAlacI gene•Is not part of the lac operon.•Has its own constitutively active promoter.•The LacI protein - AKA, the lac repressor - binds to a DNA sequence, the lacO ‘operator’, adjacent to the lac promoter. lac PromlacI PromThe ability of lactose to induce lac operon transcription involves a transcription factor, i.e. the protein product of the gene lacI.LacI protein binds DNA by means of a 3-dimensional structure called the helix-turn-helix motif:•Each of two identical protein subunits binds to the DNA.•Each subunit has two a-helices joined by a short "turn". It is the so-called recognition helix that fits into the major groove of the DNA molecule.•When the dimer binds to DNA, the two recognition helices insert into two sequential turns of the groove in opposite orientations.•As a result, the sequence of the DNA binding site is an inverted repeat consisting of two symmetric "half-sites".subunitsturnrecognition helixWatson, Fig. 18-7 & 18-11The binding sites for RNA polymerase and LacI repressor are so close that these 2 proteins cannot bind at the same time.Thus, for the lac operon, transcription and repres-sion are mutually exclusive states.activator-10 and -35 elements of the lac promoter-10 and -35 elements of the lac promoterBinding site for lac repressorBinding site for lac repressorWatson, Fig. 18-8Induction of the lac operon results from a loss of repression by LacI.REPRESSIONNO REPRESSION (= induction)When lactose is abundant, RNA polymerase is able to engage with the lac promoter and transcription is favored.TranscriptionRepressionWhen lactose is absent, the LacI repressor binds the lacO operator effectively and repression is favored.Lactose absentWhen lactose is present, an inducer molecule (I) binds to the Lac Repressor protein (R) and prevents it from binding DNA.AllosteryA chemical property of certain proteins:•Protein function is turned on or off by the binding of a particular ligand molecule.•The ligand-binding site is distant from the protein’s active site, i.e. the change in function results from a global alteration in protein structure.In the case of LacI, ligand-binding reduces the chemical affinity of the protein’s DNA binding domain.When lactose is present in the medium, it passes through the bacterial membrane into the cytoplasm. Some of this lactose is there converted into the isomer allolactose. It is allolactose which functions as the inducer by physically binding to the LacI protein.Watson, Fig. 18-13It is the presence of lactose extracellularly that induces lac operon transcription, but lactose itself is not the inducer molecule.In bacteria, the essential first step in transcription is that RNA polymerase holoenzyme must bind the core promoter.The lac operon has a weak promoter: i.e. the affinity of RNA polymerase for its DNA sequence is low, and as a consequence the rate of basal transcription (RNAP alone) is low.consensus promoter: lac operon promoter:TTGACA .... TATAATTTTACA .... TATGTT -35 -10The weak nature of the lac promoter allows LacI repression to be highly efficient:PROMOTER lacORNA PolymeraseLacILOW AFFINITYHIGH AFFINITYPROMOTER lacORNA PolymerasePROMOTER lacOHIGH OCCUPANCY RATELOW