Microbial Regulatory Systems PDF

PowerPoint® Lecture Presentations CHAPTER 6 Microbial Regulatory Systems © 2018 Pearson Education, Inc. C T A A G G T C DNA Replication A T C G T A G C Transcription A A C G C A G T T A G C C G G T A C A T A C T T G A G C T T Dark green strand is template for RNA synthesis. C C G...

PowerPoint® Lecture Presentations CHAPTER 6 Microbial Regulatory Systems © 2018 Pearson Education, Inc. C T A A G G T C DNA Replication A T C G T A G C Transcription A A C G C A G T T A G C C G G T A C A T A C T T G A G C T T Dark green strand is template for RNA synthesis. C C G T G A G C T T DNA polymerase 5′ T A 3′ C A C T G T G G C A C T G A A T G G C mRNA C G A C U U U C U C C T G A G C A A G G A G A G C C C T T C 5′ A 5′ G A C U G C U G G A G G C C U G A U U G G U Ribosome 3′ G C RNA polymerase A Translation mRNA G C G T C A C G G U Protein G A G G C Messenger RNA is template for protein synthesis. tRNA G A C C A G C 3′ 5′ Major Modes of Regulation • Gene expression: transcription of DNA into mRNA, followed by translation of mRNA into protein • Some proteins are needed in the cell at the same level all the time & are expressed constitutively, but more often proteins are needed under some conditions but not others • To regulate protein function, the cell can control • The activity of the protein • Post-translational modification (rapid) • The amount of the protein, predominant in bacteria at the levelmechanism of: • Transcription • Translation Gene organization • In bacteria, genes are often clustered into operons • Multiple genes transcribed into a single mRNA & under the control of a single regulatory site • Often encode the proteins necessary to perform coordinated function, such as biosynthesis of a given amino acid. • RNA that is transcribed from a prokaryotic operon is polycistronic • Multiple proteins encoded in a single transcript • Promoter = site of RNA polymerase binding; Terminator = end of transcription Negative control • A mechanism for regulating gene expression, in which a repressor protein prevents the transcription of genes • Binds to a specific site on DNA near the promoter, called the operator • Blocks RNA polymerase • Repressor proteins are allosteric • Conformation of the repressor is altered when bound to a small molecule called an effector • Two types of negative control: • Repression • Induction Repression • Prevention of the synthesis of an enzyme in response to a signal • Usually the presence of the final product of a biosynthetic pathway represses expression of the enzymes of the pathway • e.g. Enzymes needed to synthesize the amino acid arginine are made only when arginine is absent • Excess arginine decreases synthesis Mechanism of repression • Effector molecule (e.g. arginine) is a co-repressor that binds to the repressor and alters its conformation so that it is active & can bind to DNA Induction • Production of an enzyme in response to a signal • Usually the presence of the substrate for the enzyme • e.g. The presence of lactose induces expression of βgalactosidase, which cleaves lactose into glucose & galactose Mechanism of induction • Effector molecule (e.g. lactose) is an inducer that binds to the repressor and alters its conformation so that it is inactive and can no longer bind to DNA Positive control • A regulatory protein called an activator binds to an activator binding site near the promoter & helps RNA polymerase bind to the DNA • Promoters under positive control only weakly bind RNA polymerase without activator • Activator protein helps RNA pol recognize promoter & begin transcription • Mechanism of activator function: • Bending DNA to increase RNA polymerase binding • Interacting directly with RNA polymerase Positive control of maltose operon Activator binding sites • Activator binding sites may be near the promoter or further upstream • Looping allows activator & RNA pol to interact Regulons • Genes for maltose utilization are spread out over the chromosome in several operons • Each operon has an activator-binding site • Multiple operons controlled by the same regulatory protein are called a regulon • Regulons also exist for negatively controlled systems • Promoters may be under positive control, negative control, or multiple types of control • Some operons have multiple promoters, each with its own control system A closer look at lactose • Lactose = glucose + galactose • Transported into the cell by lactose permease • Converted into monosaccharide subunits by β- galactosidase The Lac operon lac regulatory gene DNA lacI C P O lac Structural genes lacZ • LacI encodes a repressor protein • Operon: All genes transcribed from one promoter • LacZ encodes β-galactosidase • LacY encodes lactose permease • Ensures lactose uptake & breakdown occur together • Gene expression of operon controlled by: • Promoter (P) – binds RNA polymerase • Operator (O) – binds repressor • CAP site (C) – binds activator lacY lacA Global Control and the lac Operon • Regulatory mechanisms that respond to environmental signals by regulating the expression of many different genes are called global control systems • Catabolite repression is a mechanism of global control • Controls use of carbon sources if more than one is present • e.g. The presence of glucose represses the synthesis of enzymes needed for the breakdown of other carbon sources (e.g. lactose, maltose) • Global control because many catabolic operons are affected • Ensures that the "best" carbon and energy source is used first Global Control and the lac Operon • Catabolite repression is controlled by an activator protein and is a form of positive control • Cyclic AMP receptor protein (CRP) is the activator protein • Recruits RNA polymerase to the promoter of catabolic operons • Allosteric activator: Binds to DNA only after binding of cyclic AMP • Glucose lowers cyclic AMP levels • Inhibits cyclic AMP synthesis & stimulates transport out of the cell • Therefore when glucose levels are high, cyclic AMP is low and CRP is inactive • No activation of catabolic operons Regulation of the lac Operon by the lac Repressor 18 Lac Operon • Lac repressor is coded by LacI & will bind to operator unless inducer (= lactose) is present • CRP bound to cAMP (= no glucose) binds to activator site & recruits RNA polymerase to the promoter • Expression of Lac genes only in absence of glucose & presence of lactose = Lactose © 2008 W.W. Norton & Company, Inc. MICROBIOLOGY 1/e 20 Diauxic Growth • A consequence of catabolite repression • Two exponential growth phases due to the presence of two usable energy sources Two-Component Regulatory Systems • Prokaryotes regulate cellular metabolism in response to many different environmental fluctuations • Requires mechanism to receive signals from the environment & transmit signal to specific target that to be regulated • Small molecule may enter cell & directly function as an effector • Alternatively, the external signal is detected by a sensor and transmitted to regulatory machinery (= signal transduction) • Most signal transduction systems are two-component regulatory systems • Consist of a sensor kinase protein in the membrane and a response regulator protein in the cytoplasm Two-component regulatory system • Sensor kinase detects a signal from the environment and phosphorylates itself using ATP • Phosphate is transferred to the response regulator • Typically a DNA-binding protein • Regulates transcription in either a positive or negative way • Regulatory systems have a feedback loop to terminate the response • Phosphatase removes phosphate from response regulator at a constant rate Quorum Sensing • Prokaryotes can respond to the presence of other cells of the same species • Quorum sensing: Regulatory system that monitors population size & controls gene expression based on cell density • Ensures that a sufficient number of cells are present before initiating a response that requires a certain cell density to be effective • Each species of bacterium produces a specific autoinducer molecule • Diffuses freely across the cell envelope • Reaches high concentrations inside cell only if many cells are nearby • Binds to specific activator protein and triggers transcription of specific genes Quorum Sensing • Acyl homoserine lactones (AHLs) = first autoinducer to be identified Quorum Sensing & Virulence • In pathogenic bacteria, quorum sensing can induce expression of virulence factors • Proteins that enhance invasiveness by promoting infection • e.g. Foodborne pathogenic Escherichia coli O157:H7 • Produces an AHL called AI-3 • As E. coli population increases, AI-3 produced by E coli increases & stress hormones epinephrine and norepinephrine produced by intestinal cells increases • All three signal molecules bind to receptors in E. coli membrane • Results in phosphorylation and activation of transcriptional regulators that activate toxin production Sigma Factors • A subunit of RNA polymerase called σ (sigma) interacts with consensus sequences at the promoter • Major sigma factor σ70 binds to most genes through interaction with sequences at -35 and -10 Alternative sigma factors • Some alternative sigma factors exist that recognize different consensus sequences • Heat-shock and stress response – s32 • Flagellar synthesis – s28 • Nitrogen assimilation – s54 • Allows for expression of different gene families by regulating the presence or absence of a corresponding sigma factor • = Mechanism of global gene expression control • e.g. Sporulation in B. subtilis is controlled by a set of four sigma factors Sporulation in Bacillus • Endospore formation triggered by adverse external conditions • Environmental conditions monitored by five sensor kinases • Relay system from multiple adverse conditions results in phosphorylation of sporulation factor Spo0A • High phosphorylation of Spo0A triggers sporulation • Controls expression of several genes • Leads to liberation & activation of sigma factor σF • Cascade of gene expression changes & sigma factors that control differentiation • Releases a toxin that lyses nearby cells that are not yet undergoing sporulation • Used for nutrient source • End result is formation and release of mature endospore External signals for sporulation desiccation cell density starvation P Spo0A SpoIIAB Phosphate removed by SpoIIE. Inactive SpoIIAA σF Signal from endospore activates σE; early endospore genes are transcribed. SpoIIE σF is inactive when bound to SpoIIAB. Active SpoIIAA Signal from mother cell triggers synthesis of σG in endospore and pro-σK in mother cell. Signal from endospore activates σK. Developing endospore σF σF σG σG SpoIIAB SpoIIAA binds SpoIIAB. σF is released. σE pro-σE Mother cell pro-σK σK