Lecture 13 - The lac, ara & trp Operons (1)

Biol 366, Lecture 13 Regulation of gene expression (bacterial lac, ara, trp operons) Text Section: 20.1.1 – 20.1.5 Text questions: 1 – 4 , 6, 8-9. Key terms (not exclusive): Inducer, catabolite repression, transcription attenuation, leader sequence, terminator, leader peptide...

Biol 366, Lecture 13 Regulation of gene expression (bacterial lac, ara, trp operons) Text Section: 20.1.1 – 20.1.5 Text questions: 1 – 4 , 6, 8-9. Key terms (not exclusive): Inducer, catabolite repression, transcription attenuation, leader sequence, terminator, leader peptide 1 The lactose (lac) operon of E. coli Proteins encoded by the lc operon mediate lactose metabolism Includes three genes; LacA, LacY, LacZ Includes an operator that has 3 operator sequences, O1, O2, O3 Includes a repressor, which is: - transcribed separately - constitutively expressed - represses the operon by binding the operator region (Fig 20-5) 2  The lactose (lac) operon of E. coli The Lac repressor is a tetramer, made of two homodimers Each homodimer can bind one operator sequence The Lac repressor bind to O1, and either O2 or O3 to repress transcription The Lac repressor has a “helix-turn-helix” motif in its DNA-binding domain Note. Even when the operon is repressed, a few molecules of LacZ and LacY proteins are made. These help initiate lactose metabolism when it is available. 3 lacZ protein: Is encoded by the lacZ gene Converts lactose is to galactose and glucose, and a small amount of allolactose Allolactose is the inducer of lac operon 4 The lac operon encodes for three proteins, lac A, lac Y and lac Z, required for lactose metabolism in E. coli cells. lacY protein: Is membrane-bound proteins Is a permease It mediates entry of lactose into the cell lac A protein: Is the least studies protein Is a thiogalactoside transacetylase It modifies toxic galactosides that are imported along with lactose, facilitating their removal from cells. 5  Regulation of the lac operon by repressor & inducer. When lactose is present: - Allolactose is produced - Allolactose binds the Lac repressor, inducing conformational changes - The repressor then dissociates from the operator Fig 20-6 - RNA polymerase can then initiate transcription. 6 IPTG acts like allolactose and is an effector of the lac operon IPTG: Isopropyl β-D-1-thiogalactopyranoside IPTG can bind the Lac repressor and cause its dissociation from the operator IPTG induces transcription of the lac operon. IPTG is used to induce expression of the Lac operon in most cloning / expression vectors FIG 20-6 7 LacZ recognizes X-Gal as a substrate (X-Gal): β-galactoside 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside Digestion of X-Gal by LacZ yields 5-bromo-4-chloro-3-hydroxyindole 5-bromo-4-chloro-3-hydroxyindole forms an insoluble dimer, which precipitates and produces a blue color 8 Utility of the lac operon components in molecular biology. When cloning a DNA fragment in pUC19, the LacZ gene helps select recombinant vector containing the DNA insert. How? 9 pUC18, pUC19 DNA | Thermo Scientificwww.thermoscientificbio.com Blue-white selection using pUC vectors Following cloning, vectors that took in the DNA of interest must be selected. Sometimes the vector does not contain the “insert” due to: i. Contamination with the undigested vector ii. Vector re-ligating to itself. 10 Blue-white selection using pUC vectors Blue-white selection allows selecting for bacteria that harbor the vector WITH insert. Transformed bacteria are grown on medium containing IPTG and X-Gal, and Ampicillin. WHY???? Bacteria harboring pUC vector containing the insert DNA remain white. Why? Bacteria harboring the empty pUC vector (i.e., no insert DNA) turn blue. Why? 11 Regulation of the lac operon via catabolite repression; interaction of positive and negative regulatory elements. Glucose is metabolized directly by glycolysis and is E. coli’s preferred energy source. When glucose is available, expression of the genes required for metabolizing other sugars (e.g., lactose, arabinose, etc.) is (at least partially) BLOCKED even when these sugars are also present. This is known as known as catabolite repression In the absence of glucose cell’s increase the expression of genes that allow the use of alternative food sources. 12  Regulation via catabolite repression: The effect of glucose on expression of the lac genes is mediated by two elements: Cyclic AMP (cAMP), a small-molecule effector cAMP receptor protein, or CRP, an Activator protein that bind both DNA and cAMP simultaneously. Note: When glucose is present, [cAMP] is low, and visa versa; why? When cells use glucose, the cAMP-producing enzyme, adenylate cyclase, is inhibited by byproducts of glucose metabolism. 13 When [glucose] is high & [lactose] is low: Cells use glucose, and expression of the lac genes is BLOCKED. 14  Regulation of the lac operon by CRP Low [Glucose] & No lactose Fig. 20-8b In the absence of lactose the repressor binds the operator, blocking RNA polymerase and preventing transcription of the lac genes, regardless of cAMP- CRP-Operator interactions. 15  Regulation of the lac operon by CRP high [Glucose], Lactose present - The repressor dissociates from the operator. - However, with [cAMP] low, CRP-cAMP formation and DNA binding does not occur. - Transcription from the lac operon is week. 16  Regulation of the lac operon by CRP Low [Glucose] & lactose present Fig. 20-8d - The cAMP levels increase in response to low glucose - CRP-cAMP binds the regulatory region - RNA Pol robustly binds and transcription from the lac operon is much stronger 17 Regulation via catabolite repression (a few notes) The positive and negative regulatory systems act in concert to regulate the lac operon. With no lactose, expression from the Lac operon is at background level (off) With glucose and lactose BOTH present, expression of Lac operon is low When glucose is absent, CRP-cAMP binds to a site near the Lac promoter and stimulates RNA transcription fifty-fold. Notes: The Lac repressor is a negative regulatory factor responsive to lactose levels. CRP-cAMP is thus a positive regulatory factor responsive to glucose levels 18 Regulation of the ara Operon 19  Regulation of the ara operon. Key players involved in the regulation of the ara operon. Ara genes (araB, araA and araD), are required for arabinose metabolism AraC protein is a regulatory protein araO2 (and araO1 - not shown); binding site of AraC as a repressor araI1 & araI2; binding site of AraC as activator/inducer cAMP/CRP RNA Pol Fig 20-9b. 20  Regulation of the ara operon. CRP interacts with an activator/repressor to control transcription. Positive and negative regulation by the same molecule, the AraC protein. Fig 20-9a. (a) When arabinose is absent and glucose is present (i.e. cAMP is low): AraC protein forms a dimer in which one monomer binds to araO2 and the other to araI1, forming a DNA loop. This prevents RNA polymerase from binding and transcription from the ara operon 21  Regulation of the ara operon. CRP interacts with an activators / repressors to control transcription. (b) When arabinose is present and glucose is not (in which case cAMP is high) Note: Arabinose is an AraC binds arabinose, and then activates the ara operon. “effector” for AraC. The AraC dimer changes conformation such that one monomer binds araI1 and the other binds araI2. Binding of AraC to araI2 recruits RNA polymerase to the promoter and activates transcription of the ara operon. With no glucose, CRP-cAMP also binds it binding site to help activate the ara operon. Fig 20-9b. 22 Regulation of the trp Operon 23 Regulation of the trp Operon; fine-tuning of gene expression level The trp operon encodes five genes that control synthesis of tryptophan The trp operon is regulated by a repressor system and attenuation. Fig 20-11 (a) (Text) Reference: The tryptophan pathway genes of the Sargasso Sea metagenome: New operon structures and the prevalence of non-operon organization Genome Biology 9(1):R20DOI: 10.1186/gb-2008-9-1-r20 24 A: Regulation of the trp Operon through a repressor The Trp repressor is a protein that acts as a homodimer In the absence of tryptophan: - The Trp repressor cannot bind the operator - Transcription from the trp operon is active - Trp biosynthetic enzymes are expressed, and Trp is produced Fig 20-11 (a) 25  Regulation of the trp Operon through a “repressor” When Trp is abundant: Expression from the trp operon is not needed Trp binds to & serves as “effector” for the Trp repressor “Trp-repressor” complex binds the operator, blocking transcription (Attenuation) Fig 20-11 (b) 26  B: Regulation of the trp Operon through Attenuation The trp mRNA leader sequence Trp mRNA includes a 5’ leader sequence containing four regulatory regions (1 – 4) “Sequence 1” is translated into the leader peptide. – The leader peptide has 14 aa, two of which are Trp. Sequences 2 and 3 can base pair to make a stem-loop structure Sequences 3 and 4 can also base-pair to form a stem-and- loop (hairpin) structure which acts as a transcriptional terminator Question: When does sequence 3 bind sequence 2 or 4???? 27  B: Regulation of the trp Operon through Attenuation i. When [tryptophan] is high [tRNATrp ] is high (there is no need to make Trp). Ribosomes translate quickly through Trp codons of sequence 1. Sequences 3 & 4 form a hairpin; RNA Pol stalls; transcription is terminated. This is attenuation 28  Attenuation of the trp operon ii. When tryptophan levels are low Trp [tRNA] is low, and ribosomes stall on Trp codons of sequence 1, allowing sequences 2 and 3 to associate to make a hairpin. Sequences 3 & 4 do not form terminator, and transcription proceeds. (no attenuation) The amount of free tryptophan available for protein synthesis thus determines at what level the trp operon is transcribed. 29  Effectiveness of qttenuation of the trp operon The expression of the trp genes can be repressed by: Up to ~70 fold by the repressor system alone up to ~700 fold by the repressor-attenuation system combined Fig 20-12c 30