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Chapter 21 Operons: Fine Control of Bacterial Transcription

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Chapter 21 Operons: Fine Control of Bacterial Transcription Chapter 21 Operons: Fine Control of Bacterial Transcription The E. coli genome contains over 3000 genes. Some of these are active all the time because their products are in constant demand. But some of them are turned off most of the time because their products are rarely needed. For example, the enzymes required for the metabolism of the sugar arabinose would be useful only when arabinose is present and when the organism’s favorite energy source, glucose, is absent. Such conditions are not common, so the genes encoding these enzymes are usually turned off. Why doesn’t the cell just leave all its genes on all the time, so the right enzymes are always there to take care of any eventuality? The reason is that gene expression is an expensive process. It takes a lot of energy to produce RNA and protein. In fact, if all of an E. coli cell’s genes were turned on all the time, production of RNAs and proteins would drain the cell of so much energy that it could not compete with more effi cient organisms. Thus, control of gene expression is essential to life. Gene regulation is the device to insure that proteins are synthesized in exactly the amounts they are needed and only when they are needed. Transcriptional regulation: gene expression is controlled by regulatory proteins Negative regulation: - A repressor protein inhibits transcription of a specific gene. - In this case, inducer (antagonist of the repressor) is needed to allow transcription. Positive regulation: - Activator works to increase the frequency of transcription of an gene (operon) Transcriptional regulation: gene expression is controlled by regulatory proteins Operons Operons and the resulting transcriptional regulation of gene expression permit prokaryotes to rapidly adapt to changes in the environment: new carbon sources, lack of an amino acid, etc. The genes involved in the catabolism of the carbon source or in the biosynthesis (anabolism) of the amino acid can be rapidly turned on or turned off. Negative control of expression of the Operon is the dominant case. An Operon is a set of genes that are expressed in unison, ie they are Coordinately expressed, together with the DNA control elements used for their expression. Usually, an Operon is a set of genes which are adjacent to each other on the chromosome and are Coordinately Expressed at the transcription level via a single mRNA molecule (polycistronic mRNA): Coordinate regulation. A negatively-acting protein called the Repressor regulates expression of the Operon by binding to an Operator DNA site near the promoter for transcription. Expression of the gene encoding the Repressor is usually NOT itself controlled by a Repressor. It then is expressed at all times, ie is constitutively expressed. This Operator binding site for the Repressor is the major type of DNA control element. Operons The genes that are Coordinately expressed are called Structural Genes and encode proteins that function enzymatically in a common process such as a biosynthetic or catabolic pathway. The genes that encode regulatory proteins such as Repressors are called Regulator Genes and are NOT usually part of the set of coordinately expressed operon genes. A small molecule (metabolite) usually interacts with the Repressor. This small molecule is either an Inducer or a Co-Repressor: An Inducer inactivates the Repressor in the Inducer-Repressor complex. A Co-Repressor activates the Repressor in the Inducer-Repressor complex. Thus binding of this small molecule to one site of Repressor alters its ability to bind Operator DNA at a second site on Repressor ... example of Allosterism NOTE: Products of Regulatory Genes can then diffuse or move to their targets and hence are Trans-acting Factors. Operators and DNA control elements are the targets and affect only the DNA to which they are attached; they are Cis-acting elements. Any protein binding site on DNA, e.g. transcription promoters and terminators, are cis-acting elements. Two general Operon Classes: Catabolic and Anabolic Pathways Catabolic Operons: The small molecule interacting with the Repressor is an Inducer, and is usually the sugar or metabolic which is broken down. Note the rationale: the operon should be expressed only when Inducer is present, to express the genes needed to break down (catabolize) the Inducer, e.g., sugar General Example: Structural genes A, B, C with Operator O near Promoter P. Regulator gene R constitutively expressing Repressor R inactivated by Inducer I, as shown in the following Figure: Two general Operon Classes: Catabolic and Anabolic Pathways Anabolic (biosynthetic) Operons: The small molecule interacting with the Repressor is a Co-Repressor, and is usually the end-product of the biosynthetic pathway, e.g., an amino acid. Note the rationale: the operon should be expressed only when Co-Repressor is absent, to express the genes needed to synthesize the Co-Repressor, e.g., amino acid General Example: Structural genes A, B, C with Operator O near Promoter P. Regulator gene R constitutively expressing Repressor R activated by Co-Repressor Co, as shown in the following Figure: 7.1 The lac Operon • The lac operon was the first operon discovered • It contains 3 genes coding for E. coli proteins that permit the bacteria to use the sugar lactose – Galactoside permease (lacY) which transports lactose into the cells b-galactosidase (lacZ) cuts the lactose into galactose and glucose – Galactoside transacetylase (lacA) whose function is unclear • All 3 genes are transcribed together producing 1 mRNA, a polycistronic message that starts from a single promoter – Each cistron, or gene, has its own ribosome binding site – Each cistron can be translated by separate ribosomes that bind independently of each other Control of the lac Operon • The lac operon is tightly controlled, using 2 types of control – Negative control, like the brake of a car, must remove the repressor from the operator - the “brake” is a protein called the lac repressor – Positive control, like the accelerator pedal of a car, an activator, additional positive factor responds to low glucose by stimulating transcription of the lac operon • Negative control indicates that the operon is turned on unless something intervenes and stops it • The off-regulation is done by the lac repressor – Product of the lacI gene – Tetramer of 4 identical polypeptides – Binds the operator just right of promoter Negative Control of the lac Operon • When the repressor binds to the operator, the operon is repressed – Operator and promoter sequence are contiguous – Repressor bound to operator prevents RNA polymerase from binding to the promoter and transcribing the operon • As long as no lactose is available, the lac operon is repressed Inducer of the lac Operon • The repressor is an allosteric protein – Binding of one molecule to the protein changes shape of a remote site on that protein – Altering its interaction with a second molecule • The inducer binds the repressor – Causing the repressor to change conformation that favors release from the operator • The inducer is allolactose, an alternative form of lactose Discovery of the Operon During the 1940s and 1950s, Jacob and Monod studied the metabolism of lactose by E. coli •Three enzyme activities / three genes were induced together by galactosides • Constitutive mutants need no induction, genes are active all the time • Created merodiploids or partial diploid bacteria carrying both wild-type (inducible) and constitutive alleles Lac+ phenotype F'lacY-lacZ+/ lacY+lacZ- F'lacY+lacZ-/ lacY-lacZ+ Lac- phenotype F'lacY-lacZ+/ lacY-lacZ+ F'lacY+lacZ-/ lacY+lacZ- (Partial diploid=meroploid) Discovery of the Operon • Using merodiploids or partial diploid bacteria carrying both wild-type and constitutive alleles distinctions could be made by determining whether the mutation was dominant or recessive • Because the repressor gene produces a repressor protein that can diffuse throughout the nucleus, it can bind to both operators in a meriploid and is called a trans-acting gene because it can act on loci on both DNA molecules • Because an operator controls only the operon on the same DNA molecule it is called a cis-acting gene Effects of Regulatory Mutations: (a) Wild-type and Mutated Repressor (b) Wild-type and Mutated Operator with Defective Binding Effects of Regulatory Mutations: (c& d) Wild-type and Mutated Operon binding Irreversibly Lac operon Constitutive mutation trans-acting factor, cis-acting element Repressor-Operator Interactions • Using a filter-binding assay, the lac repressor binds to the lac operator • A genetically defined constitutive lac operator has lower than normal affinity for the lac repressor • Sites defined by two methods as the operator are in fact the same Assaying the binding between lac operator and lac repressor. Cohn and colleagues labeled lacO-containing DNA with 32P and added increasing amounts of lac repressor. They assayed binding between repressor and operator by measuring the radioactivity attached to nitrocellulose. Only labeled DNA bound to repressor would attach to nitrocellulose. Red: repressor bound in the absence of the inducer IPTG. Blue: repressor bound in the presence of 1 mM IPTG, which prevents repressor–operator binding. The Mechanism of Repression • The repressor does not block access by RNA polymerase to the lac promoter • Polymerase and repressor can bind together to the lac promoter • Polymerase-promoter complex is in equilibrium with free polymerase and promoter lac Repressor and Dissociation of RNA Polymerase from lac Promoter • Without competitor, dissociated polymerase returns to promoter • Heparin and repressor prevent reassociation of polymerase and promoter • Repressor prevents reassociation by binding to the operator adjacent to the promoter • This blocks access to the promoter by RNA polymerase Mechanism Summary • Two competing hypotheses of mechanism for repression of the lac operon – RNA polymerase can bind to lac promoter in presence of repressor • Repressor will inhibit transition from abortive transcription to processive transcription – The repressor, by binding to the operator, blocks access by the polymerase to adjacent promoter.
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