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Download Ribosome stalls at trp codons, allowing 2+3 pairing Transcription
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Prokaryotic Gene Regulation Coordinate regulation of genes involved in similar functions Types of Control Negative Control Product of regulatory gene inhibits transcription Positive Control Product of regulatory gene enhances transcription Operon • Unit of coordinate gene expression • Includes structural genes and their adjacent regulatory elements • We will consider – Lac operon (inducible) – Ara operon (inducible) – Trp operon (repressible) Types of Operons Inducible Initial condition: OFF Inducer switches operon ON Repressible Initial condition: ON Repressor switches operon OFF Regulation of the Lac Operon Pcrp crp Pi I P O Structural Genes DNA Function Pcrp crp Pi I P O Lac Z Lac Y Lac A Lac Z Lac Y Lac A Protein Function Promoter for crp gene Gene for CAP protein Positive regulator Promoter for I gene Gene for Lac Repressor Negative regulator Promoter for Structural Genes Operator Gene for B-galactosidase Cleaves lactose Gene for Permease Lactose transport Gene for Acetylase Unknown Transcription from the Lac Operon Pcrp crp Pi I P Pol O Lac Z Lac Y Lac A Transcription Z, Y, A mRNA Translation B-galactosidase Permease Acetylase RNA polymerase binds to the promoter and produces a polycistronic mRNA from the Lac Z, Y and A genes. All three proteins are produced. Regulation of the Lac Operon: Low lactose, High glucose Pcrp crp crp mRNA Inactive CAP protein Pi I I mRNA Active repressor P Pol O Lac Z Lac Y Lac A No mRNA produced No Z, Y, A proteins produced Transcription from Pcrp and Pi is constitutive: always expressed in an unregulated fashion. Active repressor binds to operator and prevents RNA polymerase from reaching structural genes. Regulation of the Lac Operon: High lactose, High glucose Pcrp crp Pi I P Pol O Lac Z Lac Y Lac A Transcription crp mRNA I mRNA Z, Y, A mRNA Translation + Inactive CAP protein Lactose Active repressor B-galactosidase Permease Acetylase Inactive repressor Lactose (inducer) binds to the repressor and inactivates it. RNA polymerase transcribes Lac Z, Y and A at low frequency. Regulation of the Lac Operon: High lactose, Low glucose Pcrp crp Pi I P Pol O Lac Z Lac Y Lac A Transcription crp mRNA I mRNA Z, Y, A mRNA Translation + cAMP Inactive CAP protein Active CAP protein + Lactose Active repressor B-galactosidase Permease Acetylase Inactive repressor cAMP is produced when glucose levels are low. cAMP activates CAP. Active CAP binds to the promoter to increase RNA polymerase binding. RNA polymerase transcribes Lac Z, Y and A at HIGH frequency. Regulation of the Lac Operon: Low lactose, Low glucose Pcrp crp crp mRNA Pi I I mRNA P Pol O Lac Z Lac Y Lac A No mRNA produced + cAMP Inactive CAP protein Active repressor No Z, Y, A proteins produced Active CAP protein Although RNA polymerase binding is enhanced by Active CAP, the operator is blocked by active repressor. RNA polymerase cannot transcribe Z, Y and A. Summary of Lac Operon Regulation Level of Lactose Low Level of Lac Operon Glucose High Off Low Low Off High High On at low frequency High Low On at high frequency Mutations of the Lac Operon Pcrp CRP Pi I Functional genes: I+ P O P+ O+ Lac Z Z+ Lac Y Lac A Y+ A+ I+ I- Functional Repressor Trans-acting Non-functional Repressor Trans-acting Is Superrepressor (cannot bind lactose) Trans-acting Is > I+> I - The diffusible product of the I+ or IS allele can associate with an operator on the same piece of DNA (cis) or on a separate piece of DNA (trans). Mutations of the Lac Operon Pcrp CRP Pi I Functional genes: I+ P O P+ O+ Lac Z Z+ Lac Y Lac A Y+ A+ P+ P- Functional Promoter Cis-acting Non-functional Promoter Cis-acting O+ Oc Functional Operator Cis-acting Non-functional Operator (Operator Constitutive) Cis-acting O- Non-functional Operator (Operator region deleted) Cis-acting Mutations of the Lac Operon Pcrp CRP Pi I Functional genes: I+ P O P+ O+ Z+ Z- Functional B-galactosidase Y+ YA+ A- Functional Permease Lac Z Z+ Lac Y Lac A Y+ A+ Non-functional gene for B-galactosidase Non-functional gene for Permease Functional Acetylase Non-functional gene for Acetylase A mutation in one structural gene does not affect the production of proteins from the other structural genes. Lac Operon Mutations, Page 3-24 B-Galactosidase Permease No lactose Lactose No lactose Lactose I+P+O+Z+Y+ -- + -- + I-P+O+Z+Y+ I+P-O+Z+Y+ I+P+OcZ+Y+ I+P+O+Z-Y+/ I-P+O+Z+Y+ I+P+OcZ+Y-/ I+P+O+Z-Y+ I+P+OcZ-Y+/ I-P+O+Z+YIsP+O+Z+Y-/ I-P+O+Z-Y+ I+P-OcZ+Y+/ I+P+O+Z-Y- + -- + -- + -- + -- + + + + -+ + + --- + + ---- + --- + --- + --- Arabinose Operon DNA Function Protein Function Ara C Codes for C protein Positive and Negative regulator I O Ara B Ara A Initiator (promoter region) Binds C protein Operator Binds C protein Ara D Structural gene Structural gene Structural gene C Protein Exerts Positive and Negative Control of the Ara Operon Arabinose present Arabinose absent Summary of Ara Operon Regulation Level of Arabinose Level of Glucose Ara Operon Low High Off C protein bound to O and I, Inhibiting transcription Low Low Off C protein bound to O and I High High On at low frequency C protein + arabinose bound to I, enhancing transcription High Low On at high frequency C protein + arabinose bound to I and cAMP + CAP bound to I, enhancing transcription Tryptophan Operon trpA DNA Function RNA/Protein Function Trp R Gene for repressor Binds to operator to inhibit transcription P Promoter O Trp E, D, C, B, A Operator 5’ UTR (Leader) Structural genes Enzymes acting in pathway to produce tryptophan. Gene order correlates with order of reactions in pathway. Premature termination of transcription when trp levels are high Control of Trp Operon Transcription Trp Repressor is Inactive Initial State: ON Trp binding activates Repressor Final State: OFF tryptophan Features of the 5’ UTR •Contains complementary sequences that can form hairpin structures when transcribed into RNA •Codes for a stretch of U nucleotides that can act as a termination signal after a hairpin structure •Codes for several Trp codons as part of an unstable protein product Alternative RNA Structures from 5’ UTR Termination signal due to hairpin formed by 3+4 pairing followed by string of uracils No termination signal formed Formation of termination signal depends on level of tryptophan carried by tRNA in the cell. Attenuation Premature Termination of Transcription Ribosome translates trp codons, preventing 2+3 pairing 3+4 pairing forms terminator Antitermination Ribosome stalls at trp codons, allowing 2+3 pairing Transcription continues toward trp E, D, C. B, A Summary of Trp Operon Regulation Level of Tryptophan Trp Operon On Low High Trp repressor inactive Lack of attenuation leads to high rate of mRNA production Off Tryptophan + repressor = Active repressor Reduction of mRNA production by attenuation