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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Chapter 13 Regulation of Cellular Processes 1 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Two Approaches to Regulation • regulation of gene expression – transcription initiation – transcription elongation – translation • alter activity of enzymes and proteins – posttranslational • three domains of life differ in genome structure and regulatory mechanisms used 2 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Steps Leading from the Information Coded in DNA to a Functional Protein Bacteria Archaea 3 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 13.1 4 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Regulation of Transcription Initiation • replacement of degraded enzymes – constitutive genes • are housekeeping genes that are expressed continuously by the cell – inducible genes • are genes that code for inducible enzymes needed only in certain environments – such as b-Galactosidase 5 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Inducible Genes b-Galactosidase Enzyme • inducible enzyme functions in a catabolic pathway • inducible enzymes are present only when their substrate (inducer - effector molecule) is available • β-galactosidase reaction catalyzed is lactose hydrolysis into galactose and glucose 6 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 13.2 7 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Repressible Genes • enzymes that function in biosynthetic pathways are products of repressible genes • generally these enzymes are always present unless the end product in the biosynthetic pathway is available 8 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Control of Transcription Initiation by Regulatory Proteins • induction and repression occur because of the activity of regulatory proteins and DNA binding domains • these proteins either inhibit transcription (negative control) or promote transcription (positive control) 9 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Negative Transcriptional Control • binding of regulatory protein (repressor) at DNA regulatory site (operator) inhibits initiation of transcription – mRNA expression is reduced • repressor proteins – exist in active and inactive forms – inducers (substrates) and corepressors (enzymatic products) alter activity of repressor by binding 10 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 13.3(a) and (b) 11 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Positive Control • binding of a regulatory protein (activator protein) at a regulatory region on DNA (activator binding sites) promotes transcription initiation – mRNA synthesis is increased • activation – inactive protein is activated by inducer (activator protein) – active protein is inactivated by inhibitor 12 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 13.3(c) and (d) 13 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. “Decision” Process in Gene Expression • enzymes of a catabolite pathway are only needed (increased mRNA synthesis) when the preferred substrate is available • enzymes not synthesized when substrate absent • efficient use of energy and materials 14 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 13.4 15 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Operon Structure in Archaea and Bacteria • promoter and operator or activatorbinding sites, along with functionally related structural genes, are grouped together in the DNA • polycistronic mRNA is produced • regulatory proteins control gene expression 16 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Negative Control of Lactose (Lac) Operon • inducible genes – three structural genes coding for lactose uptake and metabolism – lac repressor (lacI) binds operator • inhibits transcription • enzymes normally not produced unless lactose present 17 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. lac Operon Figure 13.5 18 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. lac Repressor • tetramers of repressor form and bind to three operator sites – O1, O2, O3 • bend DNA and prevent RNA polymerase from accessing promoter • presence of allolactose binds repressor – no longer binds operator 19 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 13.6 20 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Regulation of the lac Operon by the lac Repressor Figure 13.7 21 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Positive Control of the lac Operon • regulated by catabolite activator protein (CAP) – regulates in response to presence or absence of glucose – allows for preferential use of glucose 22 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. The Tryptophan (trp) Operon • consists of 5 structural genes which code for enzymes needed to synthesize tryptophan • negative transcriptional control of repressible genes by trp repressor • the operon functions only in the absence of tryptophan 23 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Regulation of the trp Operon by Tryptophan and the trp Repressor Figure 13.8 24 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. The Arabinose (ara) Operon • transcriptional control by a protein (AraC) that acts both positively and negatively – activity depends on environmental conditions – inactive when arabinose present – active when arabinose absent 25 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Positive and Negative Control of ara Operon by AraC • Figure 13.9 26 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Two-Component Regulatory Systems and Phosphorelay Systems • many genes and operons are turned on or off in response to environmental conditions – the regulatory proteins involved are part of a two-component signal system which links external events to regulation of gene expression 27 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Two Component Regulatory System • found in all three domains of life • two proteins govern pathway – sensor kinase • extracellular receptor for metabolite • intracellular communication pathway – response-regulator protein • activated by sensor kinase • DNA binding protein – activator – enhances transcription needed – repressor – inhibits transcription unless needed 28 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Regulation of Porin Proteins by a Two Component Signal Transduction System Figure 13.10 29 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Phosphorelay System of Porin Proteins • Env Z (sensor kinase) – autophosphorylates in high osmolarity • OmpR (response regulator) – phosphorylated and regulates transcription • Regulate expression of porin proteins (OmpC and OmpF) depending on osmolarity 30 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Porin Protein Functions • OmpC is smaller porin protein – dominant when E. coli is in high osmolarity intestinal tract – lower levels of diffusion • Omp F is larger porin protein – dominant when E. coli is in dilute environment – allows more diffusion of solutes 31 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Regulation of Transcription Elongation • transcription can also be regulated by controlling transcription termination • this type of regulation, called attenuation, was first demonstrated with trp operon • more recently riboswitches have been demonstrated to also play a regulatory role 32 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Trp Operon Attenuation • in addition to transcription initiation control, transcription continuation is also controlled in this operon • attenuation is termination of transcription within the leader region (leader peptide) • occurs through stem-loop structures in the mRNA depending on trp level 33 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Attenuation of the trp Operon Figure 13.11 34 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Riboswitches (Sensory RNAs) • a specialized form of transcription attenuation • folding of mRNA leader sequence (the riboswitch) determines if transcription will continue or be terminated • folding pattern altered in response to mRNA binding of an effector molecule • riboswitches in gram-positive bacteria function in transcriptional termination 35 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Riboswitch of the Riboflavin (rib) Operon of Bacillus subtilis Figure 13.12 36 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Regulation of Gene Expression by Riboswitches Table 13.1 37 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Regulation of Translation • riboswitches in gram-negative bacteria regulate translation of mRNA – effector binding elements at 5’ end alters mRNA leader folding pattern • translation initiation can also be controlled by some small RNA molecules 38 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Regulation of Translation by a Riboswitch Figure 13.13 39 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Regulation of Translation by Small RNA Molecules • small (sRNAs) or noncoding (ncRNAs) RNAs – do not function as mRNA, tRNA, or rRNA – some (antisense RNAs) are complementary to mRNA and function by base pairing – may inhibit or enhance translation 40 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Translation Regulation by Antisense RNA Figure 13.14 41 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Regulation of Gene Expression by Small Regulatory RNAs Table 12.2 42 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Posttranslational Regulation • regulatory enzymes control chemotaxis proteins by – phosphorelay system and – covalent modification • irreversible (proteolysis) • reversible – methylation/demethylation – phosphorylation/dephosphorylation 43 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Chemotaxis in E. coli • Methyl-accepting chemotaxis proteins (MCPs) are chemoreceptors in membrane – binds environmental chemicals – initiates a series of interactions with cytoplasmic proteins that affects flagellar rotation • activates sensor kinase CheA (with help of CheW) which autophosphorylates • CheA phophorylates the response regulator CheY • CheY governs rotation of flagella 44 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 13.15 45 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Attractant Concentration Is High • MCP is methylated (CheR) • CheA/CheW is active (phosphorylated) • CheY is active – diffuses to flagellar motor and switches to clockwise (CW) rotation – tumbling occurs • concentration of attractant is measured every few seconds (receptors reset) 46 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Attractant Concentration Decreases • MCP is not methylated (CheB) • CheA is inactive (not phosphorylated) – CheY is inactive – flagellar rotation is counter-clockwise (CCW) and running occurs • regulatory system allows E. coli to respond to and adapt to very small amounts of attractant 47 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Methyl-Accepting Chemotaxis Proteins in E. coli Membrane Figure 13.16 48 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Global Regulatory Systems • regulatory systems that affect many genes and pathways simultaneously • important for bacteria since they must respond rapidly to a wide variety of changing environmental conditions 49 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Specialized Nomenclature • regulon – genes or operons controlled by a common regulatory protein • modulon – operon network under control of a common global regulatory protein but individual operons are controlled separately by their own regulators 50 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Mechanisms Used for Global Regulation • global regulatory systems often use many types of regulation such as: – regulatory proteins – alternative sigma factors – two component signal transduction systems – phosphorelay systems 51 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Table 13.3 52 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Catabolite Repression • regulation of transcription by both repressors and activators • diauxic growth – a biphasic growth pattern • preferential use of one carbon source over another when both are available in environment – lag occurs • after preferred substrate is exhausted followed by resumption of growth using the second source • catabolite repression plays a role in this pattern of growth 53 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 13.7 54 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Catabolite Activator Protein (CAP) • also called cyclic AMP receptor protein (CRP) • brings about the coordinate regulation of catabolite operons • exists in two forms – active form when 3’,5’-cyclic adenosine monophosphate (cAMP) is bound – inactive form when it is free of cAMP 55 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 13.18 56 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 13.19 57 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. CAP Regulation of lac Operon • all catabolite operons contain a CAP binding site • CAP must be bound to this site before RNA polymerase can bind the promoter and begin transcription 58 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Regulation of cAMP - 1 • CAP activity is modulated by cAMP • levels of cAMP controlled by adenyl cyclase (converts ATP to cAMP and PPi) by PEP – adenyl cyclase active only when little or no glucose is present – in absence of glucose, CAP is active and promotes transcription of operons used for catabolism of other sugars 59 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Regulation of the lac Operon by the lac Repressor and CAP Figure 13.20 60 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Regulation of cAMP - 2 • cAMP levels are also increased by the phosphoenolpyruvate and sugar phosphotransferase system • when glucose is available – enzyme system transfers phosphorus to glucose • when glucose is not available – enzyme system transfers phosphorus to adenyl cyclase and cAMP is made 61 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 3.21 62 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Regulation by Other Nucleotides • guanine tetraphosphate (ppGpp) – stringent response of E. coli • cyclic dimeric GMP (c-di-GMP) – regulates virulence genes – regulates transition form motile to nonmotile lifestyle in biofilm formation 63 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 13.22 64 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Stringent Response • cells are starved for amino acids • protein synthesis cannot proceed • cell decreases production of tRNA and rRNA to conserve energy – protein RelA downregulates synthesis through production of pppGpp – pppGpp and DksA destabilizes transition initiation open complexes • cell increases biosynthesis of needed amino acids 65 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 13.23 66 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 13.24 67 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Quorum Sensing • cell-to-cell communication mediated by small signaling molecules such as N-acyl-homoserine lactone (AHL) • couples cells density and intercellular communication to transcription regulation 68 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Quorum Sensing in V. fischeri • high concentrations of the AHL produced by increased density of cells diffuse back into the cell and bind to the transcriptional regulator LuxR and activates transcription • LuxR stimulates transcription of the genes for AHL synthase (luxl) and the proteins needed for light production 69 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 13.25 70 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Response to Autoinducers by V. harveyi • responds to three autoinducers – maximizes expression of bioluminescence • low cell density – low autoinducers present – LuxR not made, no bioluminescence • high cell density – any combination of inducers – LuxR made, bioluminescence occurs 71 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 13.26 72 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Sporulation in Bacillus subtilis • another example of a global regulatory system in which phosphorelay, posttranslational modification of proteins, transcription initiation regulatory proteins and alternative sigma factors play a role • starvation signal induces production of alternative sigma factors 73 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Control of Sporulation in Bacillus subtilis Figure 13.27 74 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Regulation of Gene Expression in Eukarya and Archaea • occurs at transcriptional, translational, and posttranslation levels (figure 13.1) 75 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Eukarya Gene Expression • eukarya use regulatory transcription factors to regulate transcription initiation • activator proteins bind enhancers – mRNA transcription increases • repressor proteins bind silencers – mRNA transcription decreases 76 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Eukarya Gene Expression - 2 • sRNA molecules control gene expression • antisense RNAs and micro RNAs (miRNAs) regulate translation • sRNAs also associate with spliceosome and increase alternative splicing for new proteins 77 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 13.28 78 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Regulation of Gene Expression in Archaea • most Archaea regulatory proteins function like Bacteria activators and repressors – they bind DNA sites near the promoter, enhancing or blocking the binding of RNA polymerase • some regulatory proteins function like Eukarya regulatory transcription factors by interacting with general transcription factors 79