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Chapter 16 Regulation of Gene Expression in Prokaryotes Lecture Presentation by Dr. Cindy Malone, California State University Northridge © 2015 Pearson Education, Inc. Section 16.1: Prokaryote Gene Regulation E. coli gene regulation has been studied extensively – Highly efficient genetic mechanisms have evolved that turn transcription of specific genes on and off depending on metabolic need for gene products – Respond to changes in environment – Regulate gene activity – replication, recombination, DNA repair, cell division © 2015 Pearson Education, Inc. Section 16.1: Inducible and Constitutive Enzymes Inducible enzymes – Bacteria adapt to environment by producing inducible enzymes only when specific substrates are present Constitutive enzymes – Enzymes are continuously produced regardless of chemical makeup of environment © 2015 Pearson Education, Inc. Section 16.1: Repressible System Repressible system – Presence of specific molecule inhibits gene expression – Abundance of end product in environment represses gene expression © 2015 Pearson Education, Inc. Section 16.1: Positive and Negative Control Regulation of inducible or repressible type system under positive control or negative control – Negative control: Genetic expression occurs unless shut off by regulator molecule – Positive control: Transcription occurs only when regulator molecule directly stimulates RNA production Either type of system, or both combined, can induce or repress systems © 2015 Pearson Education, Inc. 16.2 Lactose Metabolism in E. coli Is Regulated by an Inducible System © 2015 Pearson Education, Inc. Section 16.2: Lactose Metabolism Lactose: Galactose and glucose containing disaccharide – In prokaryotes, gene activity is repressed when lactose is absent and induced when available – In the presence of lactose, the concentration of enzymes responsible for its metabolism increases inducible enzymes Lactose is inducer © 2015 Pearson Education, Inc. © 2015 Pearson Education, Inc. Figure 16-1 Section 16.2: Regulatory Regions Transcription is under control of single regulatory region – In prokaryotes, genes coding for enzymes with regulatory functions are organized in clusters – Regulatory regions usually located upstream of gene cluster they control – Regulatory region on same strand cis-acting – Trans-acting elements: Molecules that bind cisacting sites © 2015 Pearson Education, Inc. Section 16.2: Cis- and Trans-Acting Sites Cis- and trans-acting sites – Regulatory site events determine if genes are transcribed into mRNA – Binding of trans-acting element at cis-acting site regulates genes cluster negatively or positively Negatively by turning off transcription Positively by turning on transcription © 2015 Pearson Education, Inc. Section 16.2: Lac Operon Lac (lactose) operon – Has three structural genes (genes coding for primary structure enzyme): lacZ, lacY, and lacA – Operon has upstream regulatory region consisting of operator and promoter (Figure 16-1) Entire gene cluster functions to provide rapid response to presence or absence of lactose © 2015 Pearson Education, Inc. © 2015 Pearson Education, Inc. Figure 16-1 Section 16.2: lacZ and -galactosidase lacZ – Encodes -galactosidase, an enzyme that converts disaccharide lactose to monosaccharides glucose and galactose – Conversion is necessary for lactose to serve as primary energy source in glycolysis © 2015 Pearson Education, Inc. Section 16.2: lacY and lacA lacY – Specifies primary structure of permease, an enzyme that facilitates entry of lactose into bacterial cell lacA – Encodes enzyme transacetylase, which may be involved in removal of toxic by-products of lactose digestion from the cell © 2015 Pearson Education, Inc. Section 16.2: Lac Operon Structural Genes Lac operon structural genes – lacZ, lacY, and lacA – All three are transcribed as single unit – Results in polycistronic mRNA (Figure 16-3) – Cistron: Part of nucleotide sequence coding for single gene – Single mRNA is translated into three gene products © 2015 Pearson Education, Inc. © 2015 Pearson Education, Inc. Figure 16-3 Section 16.2: LacI Repressor Gene Constitutive mutation lacI gene – Located close to but not part of lac operon structural genes – Produces repressor molecule, which regulates transcription of structural genes – Allosteric repressor Interacts reversibly with another molecule Causes conformational change in three-dimensional shape and change in chemical activity © 2015 Pearson Education, Inc. Section 16.2: Negative Control Lac operon: Negative control – Operon subject to negative control: Transcription occurs only when repressor fails to bind operator region (Figure 16-5) – Repressor normally binds DNA sequence in operator region Inhibits RNA polymerase Represses transcription of structural genes © 2015 Pearson Education, Inc. © 2015 Pearson Education, Inc. Figure 16-5 © 2015 Pearson Education, Inc. Figure 16-5a © 2015 Pearson Education, Inc. Figure 16-5b © 2015 Pearson Education, Inc. Figure 16-5c Section 16.2: Summary of Operon Model Summary of operon model – series of molecular interactions between proteins, inducers, and DNA – No lactose: Enzymes are not needed and expression of genes encoding enzymes is repressed – Lactose present: Indirectly induces activation of genes by binding repressor – All lactose metabolized: None is available to bind to repressor and transcription is repressed © 2015 Pearson Education, Inc. 16.3 The Catabolite-Activating Protein (CAP) Exerts Positive Control over the lac Operon © 2015 Pearson Education, Inc. Section 16.3: CAP CAP: Catabolite-activating protein – Exerts positive control over lac operon – Diminishes expression of operon when glucose present (catabolite repression) – Binds to CAP-binding site, facilitating RNA polymerase binding at promoter and facilitating transcription (Figure 16-8) © 2015 Pearson Education, Inc. Section 16.3: Transcription Levels Maximum transcription of structural genes – Repressor must be bound by lactose Does not repress operon expression – CAP must be bound to CAP-binding site © 2015 Pearson Education, Inc. © 2015 Pearson Education, Inc. Figure 16-8 © 2015 Pearson Education, Inc. Figure 16-8a © 2015 Pearson Education, Inc. Figure 16-8b Section 16.3: Glucose Inhibits CAP Binding cAMP: Cyclic adenosine monophosphate – To bind to promoter, CAP must be bound to cyclic adenosine monophosphate (cAMP) – Glucose inhibits activity of adenylyl cyclase, which catalyzes conversion of ATP to cAMP – Prevents CAP from binding when glucose is present (Figure 16-9 and Figure 16-8) © 2015 Pearson Education, Inc. © 2015 Pearson Education, Inc. Figure 16-8 © 2015 Pearson Education, Inc. Figure 16-9 16.4 Crystal Structure Analysis of Repressor Complexes Has Confirmed the Operon Model © 2015 Pearson Education, Inc. Section 16.4: Lac Operon Regulatory Regions Lac operon and regulatory regions – Detailed structure of lac operon and its regulatory regions reveals three sites for repressor binding (Figure 16-10) © 2015 Pearson Education, Inc. © 2015 Pearson Education, Inc. Figure 16-10 Section 16.4: Crystal Structure Analysis Crystal structure analysis – Determination of the crystal structure of lac repressor, repressor bound to inducer and operator DNA – Genetic and biochemical data complement missing structural interpretation In vivo, all three operators must be bound for maximum repression (Figure 16-11a, b) © 2015 Pearson Education, Inc. © 2015 Pearson Education, Inc. Figure 16-11 © 2015 Pearson Education, Inc. Figure 16-11a © 2015 Pearson Education, Inc. Figure 16-11b Section 16.4: Repression Loop Repression loop – Binding of repressor to operators O1 and O3 creates repression loop – Prevents access of RNA polymerase to promoter (Figure 16-11c) © 2015 Pearson Education, Inc. © 2015 Pearson Education, Inc. Figure 16-11c 16.5 The Tryptophan (trp) Operon in E. coli Is a Repressible Gene System © 2015 Pearson Education, Inc. Section 16.5: Tryptophan (trp) Operon Tryptophan (trp) operon – Repressible gene system in E. coli – Wild-type E. coli produce enzymes for biosynthesis of amino acids and essential macromolecules – Tryptophan present: Enzymes necessary for synthesis of tryptophan are not produced © 2015 Pearson Education, Inc. Section 16.5: Structural Genes of trp Operon Structural genes of trp operon Five contiguous structural genes transcribed as polycistronic message – Involved in tryptophan production trpE, D, C, B, A – Enzymes catalyze biosynthesis of tryptophan (Figure 16-12) trpP: promoter – binding site for RNA Pol trpO: operator – binds repressor © 2015 Pearson Education, Inc. © 2015 Pearson Education, Inc. Figure 16-12 © 2015 Pearson Education, Inc. Figure 16-12a © 2015 Pearson Education, Inc. Figure 16-12b © 2015 Pearson Education, Inc. Figure 16-12c 16.6 Alterations to RNA Secondary Structure Contribute to Prokaryotic Gene Regulation © 2015 Pearson Education, Inc. Section 16.6: Regulation by RNA’s Structure – Attenuation RNA’s secondary structure provides regulation: Attenuation and riboswitches – Attenuation: trp structural genes are preceded by leader sequence containing regulatory site called an attenuator – Transcription of leader region occurs even when operon is repressed in presence of tryptophan (attenuation) © 2015 Pearson Education, Inc. Section 16.6: Attenuation: Hairpins mRNA has potential to fold into two different stem-loops (hairpins) Antiterminator hairpin: In absence of tryptophan Terminator hairpin: In presence of tryptophan (Figure 16-13) © 2015 Pearson Education, Inc. © 2015 Pearson Education, Inc. Figure 16-13 © 2015 Pearson Education, Inc. Figure 16-13a © 2015 Pearson Education, Inc. Figure 16-13b © 2015 Pearson Education, Inc. Figure 16-13c Section 16.6: Attenuation Mechanism Leader region – Contains two tryptophan codons Antiterminator hairpin structure – Forms in absence of tryptophan; ribosome stalls at codons Terminator hairpin – Forms in presence of tryptophan; ribosome proceeds through sequence © 2015 Pearson Education, Inc. 16.7 The ara Operon Is Controlled by a Regulator Protein That Exerts Both Positive and Negative Control © 2015 Pearson Education, Inc. Section 16.7: Arabinose Operon Arabinose (ara) operon – Inducible operon analyzed in E. coli – Positive and negative regulation by AraC protein – Metabolism of arabinose (sugar) governed by enzymatic products of three structural genes, araB, A, and D (Figure 16-15a) © 2015 Pearson Education, Inc. © 2015 Pearson Education, Inc. Figure 16-15a Section 16.7: AraC Protein AraC protein – System is induced when I region is the only region bound by AraC – Transcription controlled by regulatory protein AraC, which interacts with two regulatory regions: araI and araO2 – In the presence of arabinose: AraC binds to araI and CAP–cAMP to induce expression – In the absence of arabinose and CAP–cAMP: AraC binds to both araI and araO2; forms loop and causes repression (Figure 16-15) © 2015 Pearson Education, Inc. © 2015 Pearson Education, Inc. Figure 16-15 © 2015 Pearson Education, Inc. Figure 16-15b © 2015 Pearson Education, Inc. Figure 16-15c