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Chapter 15. Regulation of Gene Expression RNA Polymerase is the Key Enzyme for Transcription • RNA polymerase involved in three phases of transcription. 1) Initiation : binds to promoter, unwinds DNA, begins polymerization of bases complementary to DNA template. 2) Elongation : movement away from promoter sigma subunit released, polymerization. 3) Termination : signal reached by RNA polymerase. - Rho dependent termination : Rho factor recognizes sequence in mRNA, binds to it, and pulls it away from RNA polymerase. - Rho independent termination : stem loop structure formed by sequence of 20 bases with a run of 6 or more U’s signals release of RNA polymerase. The Regulation of Gene Expression Can Occur at Many Steps • Binding of RNA polymerase to promoter • Shift from initiation to elongation • Release of mRNA at termination • Posttranscriptional stability of mRNA • Efficiency of ribosomes to recognize translation initiation sites • Stability of polypeptide product The Utilization of Lactose by E. coli: A Model System for Gene Regulation • The presence of lactose induces expression of the genes required for lactose utilization. – Induction : stimulation of protein synthesis – Inducer : molecule that stimulates synthesis – Lactose : inducer of genes for lactose utilization • 1950s and 1960s – Golden era of bacterial genetics • Advantages of E. coli and lactose utilization system : 1) Culture large numbers of bacteria allow isolation of rare mutants 2) Lactose genes not essential for survival (can use glucose as carbon source) 3) Induction increases expression 1000 fold making mutant identification easy Coordinate Repression and Induction of Three Genes Revealed by Studies of LactoseUtilization Mutants • Jacques Monod and Francois Jacob (Pasteur Institute in Paris) : Proposed ‘Operon Theory’ of gene regulation. 1) Single signal can simultaneously regulate expression of several genes that are clustered together on a chromosome and involve the same process. 2) Because genes are clustered, they are transcribed together as single mRNA. 3) Clusters of genes are called operons. Complementation Analysis of Mutants Identifies Lactose Utilization Genes • Monod et al. isolated many lac- mutants unable to utilize lactose. • Complementation analysis identified three genes (lacZ, lacY, and lacA) in a tightly linked cluster. • In 1961, Francois Jacob and Jacques Monod proposed the operon model for the control of gene expression in bacteria. • An operon consists of three elements: – the genes that it controls, • In bacteria, the genes coding for the enzymes of a particular pathway are clustered together and transcribed (or not) as one long mRNA molecule. – a promoter region where RNA polymerase first binds, – an operator region between the promoter and the first gene which acts as an “on-off switch”. Experimental Evidence for Repressor Protein • Isolated mutant in lacI gene – Constitutive mutant : synthesized βgalactosidase and lac permease even in absence of lactose (inducer). – lacI must be a repressor : cells must need lacI protein product to prevent expression of lacY and lacZ in absence of inducer. Inducer Releases Repressor to Trigger Enzyme Synthesis • Addition of lactose inducer caused β–galactosidase synthesis to continue. • Conclusion: Inducer binds to repressor so repressor can not bind to DNA. • Allosteric effect : inducer bound to promoter changes conformation of protein so it can not bind to DNA. Repressor has Binding Domains for Operator and for the Inducer Changes in the Operator can also Affect Repressor Activity Proteins Act in trans DNA Sites Act Only in cis • Trans acting elements : can diffuse through cytoplasm and act at target DNA sites on any DNA molecule in cell. • Cis acting elements : can only influence expression of adjacent genes on same DNA molecule. Regulatory Elements that Map Near a Gene are cis-acting DNA Sequences • cis-acting elements – Promoter : very close to gene’s initiation site – Enhancer : • can lie far way from gene • can be reversed • augment or repress basel levels of transcription lac Operator cis-elements of lac Operon Regulatory Elements that Map Far from a Gene are trans-acting DNA Sequences • Genes that encode proteins that interact directly or indirectly with target genes cis-acting elements. – Known genetically as transcription factors – Identified by : • Mapping • Biochemical studies to identify proteins that bind in vitro to cis-acting elements Repressor vs. Activator Activator Model showing how the lac repressor (red) and catabolite activating protein (dark blue) bind to the lac operon promoter, creating a 93-basepair repression loop in the lac regulatory DNA. • Repressible enzymes generally function in anabolic pathways, synthesizing end products. – When the end product is present in sufficient quantities, the cell can allocate its resources to other uses. • Inducible enzymes usually function in catabolic pathways, digesting nutrients to simpler molecules. – By producing the appropriate enzymes only when the nutrient is available, the cell avoids making proteins that have nothing to do. • Both repressible and inducible operons demonstrate negative control because active repressors can only have negative effects on transcription. The Operon Theory • The players: – lacZ, lacY, lacA genes that split lactose into glucose and galactose – Promotor site to which RNA polymerase binds – cis acting operator site – trans-acting repressor that can bind to operator (encoded by lacI gene) – Inducer that prevents repressor from binding to operator A simplified overview of the genes and regulatory units involved in the control of lactose metabolism. (This region of DNA is not drawn to scale.) The components of the wild-type lac operon The gratuitous inducer isopropylthiogalactoside (IPTG). The structural genes of the lac operon are transcribed into a single polycistronic mRNA, which is translated simultaneously by several ribosomes into the three enzymes encoded by the operon. The catabolic conversion of the disaccharide lactose into its monosaccharide units, galactose and glucose. Repression • Repression – In absence of lactose, repressor binds to operator which prevents transcription. – Negative regulatory element The response of the lac operon to the absence of lactose. Induction • Induction – Lactose present • Allolactose binds to repressor. • Repressor changes shape and can not bind to operator. • RNA polymerase binds to promotor and initiates transcription of polycistronic mRNA. The response of the lac operon to the presence of lactose. Inducible synthesis • lacI+ gene encodes a diffusible element that acts in trans by binding to any operator it encounters regardless of chromosomal location. Molecular Studies Help Fill in Details of Control Mechanisms • Radioactive tag attached to lac repressor. – Repressor from lacI+ cells purified and mixed with operator DNA, cosediment occurred. – Repressor from lacI+ mixed with mutant operator DNA, no cosediment occurred. The response of the lac operon in the absence of lactose when a cell bears the OC mutation. Constitutive • • Presence of O+ plasmid does not compensate for Oc mutation on bacterial chromosome. Operator is cis acting element. The response of the lac operon in the absence of lactose when a cell bears the I- mutation. The response of the lac operon in the presence of lactose in a cell bearing the IS mutation. Noninducible • • • All operator sites (O+) eventually occupied by superrepressor. lacI supperrepressor can not bind inducer. lacIs mutant encodes a diffusible element that binds to operator regardless of chromosomal location (trans acting element). Copyright © 2010 Pearson Education, Inc. Question • For the lac genotypes shown in the accompanying table, predict whether the structural gene (Z) is constitutive, permanently repressed, or inducible in the presence of lactose. Genotype I+O+Z+ I-O+Z+ I+OCZ+ I-O+Z+/F’I+ I+OCZ+/F’O+ IsO+Z+ IsO+Z+/F’I+ Constitutive Repressed Inducible Question • For the genotypes and conditions (lactose present or absent) shown in the accompanying table, predict whether functional enzymes (F), nonfunctional enzymes (NF), or no enzymes (NO) are made. Genotype Conditions I+O+Z+ No Lactose I+OCZ+ Lactose I-O+Z- No Lactose I-O+Z- Lactose I-O+Z+/F’I+ No Lactose I+OCZ+/F’O+ Lactose I+O+Z-/F’I+O+Z+ Lactose I-O+Z-/F’I+O+Z+ No Lactose ISO+Z+/F’O+ No Lactose I+OCZ+/F’O+Z+ Lactose F NF NO Positive Control Increases Transcription of lacZ, lacY, and lacA • cAMP binds to CRP (cAMP receptor protein) when glucose is low. • CRP binds to regulatory region. • Enhances activity of RNA polymerase at the lac promoter. Positive Regulation The formation of cAMP from ATP, catalyzed by adenyl cyclase. In the absence of glucose, cAMP levels increase, resulting in the formation of a CAP-cAMP complex, which binds to the CAP site of the promoter, stimulating transcription. In the presence of glucose, cAMP levels decrease, CAP-cAMP complexes are not formed, and transcription is not stimulated. How Regulatory Proteins Interact with RNA Polymerase • Negative regulators (lac repressor) – Physically block DNA-binding sites of RNA polymerase. • Positive regulators • Establish physical contact with RNA polymerase enhancing enzyme’s ability to initiate transcription. Summary of Gene Regulation in lac Operon • The trp operon is an example of a repressible operon, one that is inhibited when a specific small molecule binds allosterically to a regulatory protein. • In contrast, an inducible operon is stimulated when a specific small molecule interacts with a regulatory protein. – In inducible operons, the regulatory protein is active (inhibitory) as synthesized, and the operon is off. – Allosteric binding by an inducer molecule makes the regulatory protein inactive, and the operon is on. Biosynthesis of Trp trp Operon The components involved in the regulation of the tryptophan operon. Trp Operator Leader RNA (a) The trp operon, showing the leader region, followed by the five trp genes. (b) The leader region in the mRNA is enlarged to show the translation start codon (AUG), the two tryptophan codons (UGGUGG), and the string of U residues at the end of the leader. The four regions marked 1, 2, 3, and 4 indicate the four sequence regions that have potential to form stems by base pairing. In the absence of tryptophan, an inactive repressor is made that cannot bind to the operator (O), thus allowing transcription to proceed. In the presence of tryptophan, it binds to the repressor, causing an allosteric transition to occur. This complex binds to the operator region, leading to repression of the operon. The Attenuation of Gene Expression: Fine Tuning of the trp Operon through Termination of Transcription • The presence of tryptophan activates a repressor of the trp operon – trpR gene produces repressor. – Corepressor : tryptophan binds to trp repressor allowing it to bind to operator DNA and inhibit transcription. Attenuation in the trp Operon Attenuator λ Phage Life Cycle ▶ Lytic Cycle: Cro ▶ Lysogenic Cycle: CI λ Phage Map Control Region of phage λ □ PR and PL are strong, constitutive promoter. □ PRM is a weak promoter and only directs efficient transcription when an activator is bound. Transcriptional Control in Lytic and Lysogenic Growth Relative Positions of Promoters and Operators ► Upon infection, transcription is immediately initiated from the two constitutive promoters PR and PL. ► PR directs synthesis of both Cro and CII. ► Cro expression favors lytic cycle. ► When Cro reaches a certain level, it will bind OR3 and block PRM. ► CII expression favors lysogenic growth by directing transcription of the repressor gene. ► For successful lysogeny, repressor must then bind to OR1 and OR2 and activate PRM before Cro can inhibit that promoter. Cooperative Binding of λ Repressor ► CI: OR1>OR2, OR3 ► Cro: OR3>OR2, OR1 Action of λ Repressor and Cro Antiterminator Genes and Promoters Involved in the Lytic/Lysogenic Choice Establishment of Lysogeny • If growth is good, FtsH (protease) is very active, CII is destroyed efficiently, repressor is not made, and phage tend to grow lytically. • In poor growth conditions, the opposite happens: low FtsH activity, slow degradation of CII, repressor accumulation, and a tendency toward lysogenic development. • Levels of CII are also modulated by the phage protein CIII. • CIII stabilizes CII, probably because it acts as an alternative substrate for FtsH. Gene Regulation in Eukaryotes In Eukaryotes Three RNA Polymerases Transcribe Different Sets of Genes • RNA polymerase I transcribes rRNA. – rRNAs are made of tandem repeats on one or more chromosomes. – RNA polymerase I transcribes one primary transcript which is broken down into 28S and 5.8S by processing. • RNA polymerase III transcribes tRNAs and other small RNAs (5s rRNA, snRNAs). • RNA polymerase II recognizes cis-acting regulatory regions composed of one promoter and one or more enhancers. – Promoter contains initiation site and TATA box. – Enhancers are distant from target gene. • Sometimes called upstream activation sites (UAS). • RNA polymerase II transcribes all protein coding genes. – Primary transcripts are processed by splicing, a poly A tail is added to the 3’ end, and a 5’ GTP cap is added. trans-acting Proteins Control Transcription from Class II Promoters • Basal factors bind to the promoter. – TBP : TATA box binding protein – TAF : TBP associated factors • RNA polymerase II binds to basal factors. Assembly of General Transcription Factors Copyright © 2010 Pearson Education, Inc. Activator Proteins • Also called transcription factors. • Bind to enhancer DNA in specific ways. • Interact with other proteins to activate and increase transcription as much as 100-fold above basal levels. • Two structural domains mediate these functions. – DNA-binding domain – Transcription-activator domain • Transcriptional activators bind to specific enhancers at specific times to increase transcriptional levels. Formation of DNA Loop Copyright © 2010 Pearson Education, Inc. Some Proteins Affect Transcription without Binding to DNA • Coactivator : binds to and affects activator protein which binds to DNA. • Enhancerosome : multimeric complex of proteins – Activators – Coactivators – Repressors – Corepressors Repressors • Reduction of transcriptional activation but do not affect basal level of transcription. – Activator-repressor competition – Quenching (corepressors) • Some repressors stop basal level of transcription. – Binding directly to promoter – Bind to DNA sequences farther from promoter, contact basal factor complex at promoter by bending DNA causing a loop where RNA polymerase can not access the promoter. Transcription Factors may Act as Activators or Repressors or Have No Affect • Action of transcription factor depends on – Cell type – Gene it is regulating Other Mechanisms of Gene Regulation • Chromatin structure – Slows transcription – Hypercondensation stops transcription • Genomic imprinting – Silences transcription selectively if inherited from one parent • Some genes are regulated after transcription – RNA splicing can regulate expression – RNA stability controls amount of gene product – mRNA editing can affect biological properties of protein – Noncoding sequences in mRNA can modulate translation – Protein modification after translation can control gene function Normal Chromatin Structure Slows Transcription Remodeling of Chromatin Mediates the Activation of Transcription Copyright © 2010 Pearson Education, Inc. Hypercondensation over Chromatin Domains Causes Transcriptional Silencing Noncoding RNAs Play Multiple Roles in Controlling Gene Expression • Only a small fraction of DNA codes for proteins, rRNA, and tRNA. • A significant amount of the genome may be transcribed into noncoding RNAs. • Noncoding RNAs regulate gene expression at two points: mRNA translation and chromatin configuration. MicroRNAs (miRNAs) and Small Interfering RNAs (siRNAs) • miRNAs – 21-25 nt, hairpin (stem-loop) structure. • siRNAs – 20-25 nt, dsRNA. Copyright © 2010 Pearson Education, Inc. Effects on mRNAs by MicroRNAs (miRNAs) and Small Interfering RNAs (siRNAs) • MicroRNAs (miRNAs) are small single-stranded RNA molecules that can bind to mRNA. • These can degrade mRNA or block its translation. Hairpin miRNA Hydrogen bond Dicer miRNA 5′ 3′ (a) Primary miRNA transcript mRNA degraded miRNAprotein complex Translation blocked (b) Generation and function of miRNAs • The phenomenon of inhibition of gene expression by RNA molecules is called RNA interference (RNAi). • RNAi is caused by small interfering RNAs (siRNAs). • siRNAs and miRNAs are similar but form from different RNA precursors. The Nobel Prize in Physiology or Medicine 2006 "for their discovery of RNA interference – gene silencing by double-stranded RNA" Mechanism of Action of RNAi