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Biol115 The Thread of Life" Lecture 10" Regulation of gene expression I: prokaryotic systems" Cheese is milk's leap toward immortality" ~Cliff Fadiman! Objectives" • Describe the organization of bacterial DNA into operons.! • Compare and contrast the operations of a repressible and an inducible operon." • Key terms: allosteric effector, allosteric protein, inducible operon, operator, operon, promoter, repressible operon, repressor" Biol115_2014_Lecture 10" 2" Principles of Biology" " " Chapter 18 Prokaryotic Gene regulation" Biol115_2014_Lecture 10" 3" Overview: conducting the genetic orchestra" • The genome contains all of the information needed for the growth and development of individuals." • Gene expression refers to the use of DNA sequences to synthesise RNA and proteins." • Some genes are expressed all the time (constitutive or housekeeping), while others are expressed only when needed (regulated)." Biol115_2014_Lecture 10" 4" Gene expression in prokaryotes Since bacteria have no nucleus, transcription and translation can occur in the same space. Biol115_2014_Lecture 10" 5" Bacteria often respond to environmental change by regulating transcription" • Natural selection has favoured bacteria that produce only the products needed by that cell" • A cell can regulate the production of enzymes by feedback inhibition or by gene regulation" • Gene expression in bacteria is controlled by the operon model" Biol115_2014_Lecture 10" 6" Tryptophan synthesis A diagram shows that tryptophan synthesis is inhibited by binding of tryptophan to an enzyme required for its synthesis. In the absence of tryptophan, E. coli activates a metabolic pathway to begin tryptophan synthesis from a precursor. When tryptophan is abundant, either from the environment or through synthesis, the metabolic pathway undergoes feedback inhibition. Tryptophan inhibits the activity of an enzyme in its synthesis pathway. Additionally, tryptophan can repress the tryptophan synthesis pathway by negatively regulating the gene expression of the enzymes necessary for tryptophan synthesis. 5 enzymes are required for tryptophan synthesis. Tryptophan inhibits both an enzyme in the pathway and the expression of the genes encoding the enzymes. Biol115_2014_Lecture 10" 7" Operons: The basic concept" • A cluster of functionally related genes can be under coordinated control by a single on-off “switch”" • An operon is the entire stretch of DNA that includes the operator, the promoter, and the genes that they control" • The regulatory “switch” is a segment of DNA called an operator usually positioned within the promoter" • The operon can be switched off by a protein repressor! • The repressor prevents gene transcription by binding to the operator and blocking RNA polymerase" " Biol115_2014_Lecture 10" 8" Operons: The basic concept" • The operon can be switched off by a protein repressor! • The repressor prevents gene transcription by binding to the operator and blocking RNA polymerase" • The repressor is the product of a separate regulatory gene! • The repressor can be in an active or inactive form, depending on the presence of other molecules" Biol115_2014_Lecture 10" http://tinyurl.com/kzpscbe" 9" Contents of an operon" "Promoter: RNA polymerase binding site" "Repressor: regulatory protein that binds DNA (see operator)" "Operator (controlling site): DNA region that is downstream of promoter and to which the repressor binds" "Coding sequences: also known as structural genes, which specify the expression of proteins involved in a specific metabolic pathway" "Terminator: DNA sequence which signifies the end of an operon." Biol115_2014_Lecture 10" 10" More definitions" • Regulator gene – A gene that codes for a product (typically protein) that controls the expression of other genes (usually at the level of transcription)." • Structural gene – A gene that codes for any RNA or protein product other than a regulator." Biol115_2014_Lecture 10" 11" The concept of operons" http://www.nature.com/scitable/content/33138/pierce_16_3_FULL.jpg" Note: for this lecture, assume that the regulator protein = repressor" Biol115_2014_Lecture 10" 12" The repressor protein – a shape shifter!" • Each operon is controlled by its own repressor protein." • Repressor proteins are called allosteric proteins. Allosteric proteins are proteins that change their shape when bound to a specific molecule called an allosteric effector." Biol115_2014_Lecture 10" 13" The trp (tryptophan) operon" • If amino acids are present in the growth medium E. coli will “import” amino acids before it makes them." • By default the trp operon is on and the genes for tryptophan synthesis are transcribed" • When tryptophan is present, it binds to the trp repressor protein, which causes it to change its shape and thus turn off the operon. " • The repressor is active only in the presence of its corepressor, tryptophan; thus the trp operon is turned off (repressed) if tryptophan levels are high" • A corepressor is a molecule that cooperates with a repressor protein to switch an operon off" Biol115_2014_Lecture 10" 14" Insert Fig. 18-3 P 355" Biol115_2014_Lecture 10" 15" Biol115_2014_Lecture 10" 16" Trp operon – genetic terminology" Element! Function! Notes! trpA-E" Genes that encodes enzymes involved in tryptophan synthesis" trpR" Not a part of the trp Gene that encodes the trp repressor protein operon, but regulates (when bound to DNA (operator), this proteins transcription of the trp prevents transcription at the trpoperon)" operon" Biol115_2014_Lecture 10" Structural genes of the trp operon" 17" Lactose metabolism" • Lactose = major sugar of milk " • Lactose is an uncommon nutrient for bacteria" • Enzymes for lactose metabolism are normally not expressed (turned off)." Biol115_2014_Lecture 10" 18" Lactose" • A disaccharide of glucose and galactose" • 3 enzymes are involved:" " " 1. permease (lacY)- " allows lactose to enter bacterial cells" 2. β-galactosidase (lacZ) - breaks down lactose to glucose and galactose" 3. transacetylase (lacA) - unknown function but assists lactose breakdown" Biol115_2014_Lecture 10" 19" The lac operon Consists of a promoter (P), operator (O) and three structural genes (lacZ, lacY and lacA). A separate gene, lacI, encodes the Lac repressor. Biol115_2014_Lecture 10" 20" The lac operon In the absence of lactose, the Lac repressor is bound to the operator, preventing RNA polymerase from transcribing the structural genes. Biol115_2014_Lecture 10" 21" The lac operon In the presence of lactose, some allolactose is produced. Allolactose binds to allosteric site of Lac repressor, changing its conformation so that it can no longer bind the operator. This allows RNA polymerase to transcribe the structural genes. Biol115_2014_Lecture 10" 22" Repressor does not completely inhibit transcription! So very small amounts of the enzymes are made! Biol115_2014_Lecture 10" 23" Lac repressor protein (violet) forms a tetramer which binds to DNA, causing a loop to form in the DNA. As a result expression of the lac operon is turned off. " Biol115_2014_Lecture 10" 24" Lac operon – genetic terminology! Element! lacZ" Function! Gene that encodes the β-galactosidase protein (enzyme that breaks down lactose to glucose and galactose)" Notes! lacY" Gene that encodes the permease protein (a Structural genes of the membrane protein that imports lactose into lac operon" bacterial cells)" lacA" Gene that encodes the transacetylase protein (unknown function)" lacI" Gene that encodes the lac repressor protein (when bound to DNA (operator), this proteins prevents transcription at the lac operon)" Biol115_2014_Lecture 10" Not a part of the lac operon, but regulates transcription of the lac operon" 25" Operon! Conditions! Repressor! Low intracellular Is inactive and does not bind to tryptophan" operator sequence on DNA" trp! Result! RNA polymerase transcribes trp operon genes, which results in tryptophan biosynthesis" High intracellular tryptophan" Is active. Tryptophan (corepressor) binds to repressor and induces the repressor protein to bind to operator sequence on DNA." No extracellular lactose" RNA is obstructed from Is active. It binds to the operator transcribing lac operon genes. sequence on DNA." No lactose metabolising enzymes are synthesised." High extracellular lactose" Is inactive. Allolactose (inducer) binds to repressor and reduces its affinity for DNA. Repressor protein falls off DNA." lac! Biol115_2014_Lecture 10" RNA polymerase is obstructed from transcribing trp operon genes. Tryptophan biosynthesis stops." RNA polymerase is free to transcribe lac operon genes. Concentration of lactose metabolising enzymes increases." 26" Repressible and inducible operons" • A repressible operon is one that is usually on; binding of a repressor to the operator shuts off transcription" • The trp operon is a repressible operon.! • An inducible operon is one that is usually off; a molecule called an inducer inactivates the repressor and turns on transcription" • The lac operon is an example of an inducible operon." Biol115_2014_Lecture 10" 27" Repressible and inducible operons" • Inducible enzymes usually function in catabolic pathways; their synthesis is induced by a chemical signal" • Repressible enzymes usually function in anabolic pathways; their synthesis is repressed by high levels of the end product" • Regulation of the trp and lac operons involves negative control of genes because operons are switched off by the active form of the repressor" Biol115_2014_Lecture 10" 28" Summary" • In inducible regulation, the gene is regulated by the presence of its substrate (the inducer) – e.g. allolactose and the lactose operon." • In repressible regulation, the gene is regulated by the product of its enzyme pathway (the corepressor) – e.g. tryptophan metabolism." Biol115_2014_Lecture 10" 29" You should now be able to:" 1. Explain the concept of an operon and the function of the operator, repressor, and co-repressor." 2. Explain the adaptive advantage of grouping bacterial genes into an operon." 3. Explain how repressible and inducible operons (using the trp and lac operons as examples) differ and how those differences reflect differences in the pathways they control." Biol115_2014_Lecture 10" 30"