1st set of Journal Clubs this Wednesday! Presenters: Your presentation MUST be on a CD for the class presentation!!!!!!!! You may GIVE ME either a CD or a floppy disk of your presentation. Make sure you include a title slide with the title of paper and your names. You also need to bring me a hard copy (printed sheets) of slides; Black & white is fine. Bind sheets somehow e.g. staple, put in binder. 1 Readers: Bring me a printout of your four questions. Put your name and student number on the top. Hand in to me at the BEGINNING of the class. 2 1 Regulation of Transcription in Prokaryotes •lac Operon •ara Operon •trp Operon 3 Operon: A group of contiguous, coordinately controlled genes. A single mRNA molecule transcribed from an operon contains multiple ORFs - called a polycistronic message. 4 2 Transcriptional Regulation of Operons •Regulatory sequences adjacent to an operon determine whether it’s transcribed or not. •Regulatory sequences are primarily ‘operators’ (repressor binding sequences). Can also include activator binding sequences. •Regulatory proteins work with regulatory sequences to control transcription of the operon. 5 Induction and Repression •Induction refers to increased expression of genes in response to a metabolite. •Repression refers to decreased expression of genes in response to a metabolite. •IPTG, the artificial inducer of the lac operon, is an example of a ‘gratuitous’ inducer: It cannot be metabolized by β-galactosidase enzyme, but it still induces β-galactosidase synthesis. 6 3 The lac operon allows the use of lactose as a carbon source for bacteria. Figure 7.1 7 Genes Encoded by the lac Operon •lacZ encodes β-galactosidase that cleaves lactose into galactose and glucose, AND converts lactose to the inducer allolactose. •lacY encodes galactoside permease which is required for transport of lactose into the cells. •lacA encodes galactoside transacetylase which transfers acetyl groups from acetyl-CoA to β-galactosides. The biological function of the enzymatic activity is unclear. 8 4 β-galactosidase Activity 1. Figure 7.2 2. β-galactosidase lactose β-1,4 linkage allolactose β-1,6 linkage Figure 7.4 9 Regulation of the lac operon Involves: Negative control: ‘Brake in car is on unless something causes it to be released.’ Positive control: ‘An activator causes the accelerator pedal to be pushed.’ Cis-acting sequence: Can only function if on same piece of DNA that its regulating Trans-acting factor: A gene product that can act in ‘trans’ i.e. diffuse to a location at a distance from where it was made. 10 5 Negative Control of the lac Operon Figure 7.3 11 Monod showed that Z+Y- mutants did not take up a radioactively labeled galactoside in the presence of inducer. He concluded that lacY must encode an enzyme responsible for transporting the galactoside into cells: called it galactoside permease. 12 6 lac Genetics I Figure 7.5 a and b 13 lac Genetics II Figure 7.5 c and d 14 7 Jacob and Monod’s Operon Hypothesis Based on Genetics 1. There are two key control elements of the operon: the repressor gene and the operator to which the repressor gene product binds. 2. There is a specific interaction between the inducer and repressor that prevents the repressor from binding to the operator. 3. All three lac genes are clustered under a single control unit. 4. Subsequent deletion analysis showed that there is a promoter necessary for the expression of all three lac genes. Biochemical studies confirm all of the tenets of 15 Jacob’s and Monod’s hypothesis. Isolation of the lac Repressor Assay for lac repressor activity: Binding to the labeled synthetic inducer IPTG. Problem: lac repressor is present in very tiny quantities in the cell. Solution: Isolate repressor from mutant E. coli strain with lacIt mutation that causes repressor to bind to IPTG more tightly than normal. 16 8 Prediction: If protein Gilbert and Müller-Hill isolated really was the lac repressor then it should bind to the lac operator in an inducer sensitive manner (i.e. the addition of inducer should prevent the repressor from binding to the operator). Experiment: Cohn and colleagues used a nitrocellulose filter binding assay. They mixed 32P-labeled lacO DNA with the protein from above, and either added or left out IPTG. See Figure 7.6. (See Figure 5.34 for reminder of how the assay works.) Result: In the presence of protein and ABSENCE of IPTG the labeled lacO DNA was retained on the filter. Conclusion: The lac repressor had been isolated. Also found the Oc operator bound with lower affinity to the repressor as compared to the WT operator. (See Figure 7.7) 17 How does the lac repressor work? 1971 - Pastan and colleagues showed that RNA Pol can bind tightly to the lac promoter and form an open promoter complex in the presence of the lac repressor. 1987 - Straney and Crothers showed that RNA Pol and the lac repressor can bind simultaneously to the lac promoter. Hypothesis: The lac repressor prevents the transition from transcription initiation to elongation. 18 9 Lee and Goldfarb Expt. Expt: Run-off transcription assay. 1. Pre-incubate labeled DNA (contains lac control region and beginning of lacZ gene) with (or without) repressor for 10 min. 2. Add RNA Pol. 3. Add heparin, and all components of transc. rxn. except CTP. 4. Add labeled [α-32P]CTP with or without IPTG. 19 Result of Lee and Goldfarb Expt. Conclusion: RNA Pol forms an open complex with the lac promoter even in the presence of the lac repressor in vitro. Observed that short abortive transcripts are even shorter in the presence of the repressor. Added further support for the idea that the repressor prevents the transition from initiation to elongation. 20 10 Problem with Lee and Goldfarb Expt: Nonphysiological concentrations of repressor, RNA Pol etc. were used. (Concentrations were higher than in vivo.) Record and Colleagues: Kinetic studies of effect of lac repressor on dissociation of RNA Pol from promoter in vitro using conditions closer to in vivo situation. • Made RNA Pol/lac promoter complexes. • Added or didn’t add heparin or lac repressor. • Measured rate of abortive transcript synthesis by including a fluorescently labeled UTP analog. Labeled pyrophosphate was released when this analog was incorporated into RNA. Got paradoxical results! 21 Result of Record and colleagues Expt. Results suggest that the lac repressor does prevent 22 binding of RNA Pol to the promoter! 11 There are 3 lac operators Figure 7.10 23 All three operators are important for repression. Figure 7.11 24 12 Structure of lac repressor tetramer bound to DNA one dimer of tetramer operator DNA 25 Positive Control of the lac Operon + CAP protein These are trans-acting factors. 26 13 Cis-acting sequence is activator (or CAP) binding site. Figure 7.16 27 cAMP Signals Low Glucose actually activator binding-site 28 14 lac Operon Off low 29 lac Operon Very Weakly On 30 15 lac Operon Fully Induced 31 CAP -cAMP Activity •CAP-cAMP complex recruits RNA Pol to the major (P1) promoter of the lac operon to form closed promoter complex •lac operon has alternative promoter (P2)that is inefficient. CAP-cAMP reduces initiation at P2 and stimulates initiation at P1. 32 16 CAP and RNA Pol bind cooperatively to DNA, and physically interact. Evidence: 1. CAP and RNA Pol cosediment in presence of cAMP. 2. When CAP and RNA Pol are both bound to lac control region, they can be chemically cross-linked. 3. DNase footprinting shows that CAP and RNA Pol bind to adjacent binding sites in DNA. 4. Certain mutations in CAP that reduce activation specifically affect what is thought to be RNA Pol binding region of CAP. (Mutations don’t affect binding of DNA by CAP). 5. Deletion of αCTD of RNA Pol, region thought to bind to CAP, prevents activation by cAMP-CAP. 6. In crystal structure, cAMP-CAP and αCTD touch when cAMP-CAP and RNA Pol both bound to DNA. 33 Interaction of CAP-cAMP, RNA Pol and DNA of lac control region. 34 17 The ara Operon •another example of operon that has both positive and negative regulation •araB, A, and D encode the 3 arabinose metabolizing enzymes •araC encodes the control protein AraC which is both a positive regulator (in the presence of arabinose) and a negative regulator (in the absence of arabinose). •cAMP-CAP complex also acts as a positive regulator 35 Organization of the ara operon Figure 7.21 a 36 18 Control of the ara Operon I - Negative Figure 7.21 b araPBAD •When arabinose is absent, the AraC protein acts as a negative regulator. •AraC acts as a dimer, and causes the DNA to loop. Looping brings the I1 and O2 sites in proximity to one another. •One AraC monomer binds to I1 and a second monomer binds to O2. •Binding of AraC prevents RNA Pol from binding to 37 the PBAD promoter Control of the ara Operon II - Positive Figure 7.21 c araPBAD •When arabinose is present, it binds to AraC and changes AraC conformation •An arabinose-AraC dimer complex binds preferentially to I1 and I2, and NOT to O2 which causes ‘opening’ of the loop. This allows RNA Pol to bind to PBAD. •If glucose levels are low, cAMP-CAP complex binds to Pc. •Active transcription occurs. 38 19 Negative Autoregulation of araC Transcription Figure 7.26 •High levels or AraC cause the protein to bind to O1 and inhibit transcription of the araC gene negative autoregulation. 39 The trp Operon •trp operon encodes anabolic enzymes that synthesize tryptophan. (Note this distinction from catabolic enzymes of lac and ara operons.) •genes for 5 enzymes are trpE, D, C, B and A •trp operon is regulated at level of: -transcription initiation -transcription termination (rho independent) -mechanism of anti-termination 40 20 Negative Regulation of the trp Operon by Repression Figure 7.27 41 Attenuation - Another Mode of Negative Control trpE, D, C, B and A are further downstream 42 21 Hairpin loops that leader-attenuator transcript can form 1-2 hairpin 3-4 attenuator hairpin alternative 2-3 hairpin 43 Defeating Attenuation when trp levels are low trpE, D, C, B and A are further downstream 44 22 Attenuation of trp operon in B. subtilus Different mechanism. Read your text: Pg 205-208 BEFORE Wednesday!! 45 23