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 O I P Lac Z Lac Y Lac A DNA Function Protein Function Pcrp Promoter for crp gene crp Gene for CAP protein Positive regulator Pi Promoter for I gene I Gene for Lac Repressor Negative regulator P Promoter for Structural Genes O Operator Lac Z Gene for B-galactosidase Cleaves lactose Lac Y Gene for Permease Lactose transport Lac A Gene for Acetylase Unknown Structural Genes

Transcription from the Lac Operon Lac Z Lac Y Lac A P O Pi I Pcrp crp RNA polymerase binds to the promoter and produces a polycistronic mRNA from the Lac Z, Y and A genes. All three proteins are produced. Pol Z, Y, A mRNA Transcription B-galactosidase Translation Permease Acetylase

Regulation of the Lac Operon: Low lactose, High glucose Lac Z Lac Y Lac A P O Pi I Pcrp crp Pol 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. Active repressor crp mRNA I mRNA Inactive CAP protein No mRNA produced No Z, Y, A proteins produced

Regulation of the Lac Operon: High lactose, High glucose Lac Z Lac Y Lac A P O Pi I Pcrp crp Active repressor crp mRNA I mRNA Inactive CAP protein Pol Z, Y, A mRNA Transcription Translation B-galactosidase Permease Acetylase Lactose Lactose (inducer) binds to the repressor and inactivates it. RNA polymerase transcribes Lac Z, Y and A at low frequency. + Inactive repressor

Regulation of the Lac Operon: High lactose, Low glucose Lac Z Lac Y Lac A P O Pi I Pcrp crp Active repressor crp mRNA I mRNA Inactive CAP protein Lactose + Pol Z, Y, A mRNA Transcription Translation B-galactosidase Permease Acetylase + 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. cAMP Active CAP protein

Regulation of the Lac Operon: Low lactose, Low glucose Active repressor Lac Z Lac Y Lac A P O Pi I Pcrp crp crp mRNA I mRNA Inactive CAP protein cAMP + Although RNA polymerase binding is enhanced by Active CAP, the operator is blocked by active repressor. RNA polymerase cannot transcribe Z, Y and A. Pol No mRNA produced No Z, Y, A proteins produced

CAP Protein Structure Allows Binding to DNA Domains are regions on a protein with specific functions; motifs are characteristic structures within a domain CAP has a DNA binding domain with a helix-turn-helix structural motif Helices fit into the major groove on DNA

Summary of Lac Operon Regulation Level of Lactose Level of Glucose Lac Operon Low High

Mutations of the Lac Operon Pcrp Pi O CRP I P Lac Z Lac Y Lac A Functional genes: I+ P+ O+ Z+ Y+ A+ I+ Functional Repressor Trans-acting I- Non-functional Repressor Is Superrepressor (cannot bind lactose) 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 Pi O CRP I P Lac Z Lac Y Lac A Functional genes: I+ P+ O+ Z+ Y+ A+ P+ Functional Promoter Cis-acting P- Non-functional Promoter O+ Functional Operator Oc Non-functional Operator (Operator Constitutive) O- (Operator region deleted)

Mutations of the Lac Operon Pcrp Pi O CRP I P Lac Z Lac Y Lac A Functional genes: I+ P+ O+ Z+ Y+ A+ Z+ Functional B-galactosidase Z- Non-functional gene for B-galactosidase Y+ Functional Permease Y- Non-functional gene for Permease A+ Functional Acetylase A- 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+Y- IsP+O+Z+Y-/ I-P+O+Z-Y+ I+P-OcZ+Y+/ I+P+O+Z-Y-

Arabinose Operon Ara C I O Ara B Ara A Ara D DNA Function Protein Function Ara C Codes for C protein Positive and Negative regulator I Initiator (promoter region) Binds C protein O Operator Ara B Structural gene Ara A Ara D

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

Tryptophan Operon Trp R P O Trp E, D, C, B, A 5’ UTR (Leader) trpA DNA Function RNA/Protein Function Trp R Gene for repressor Binds to operator to inhibit transcription P Promoter O Operator Trp E, D, C, B, A Structural genes Enzymes acting in pathway to produce tryptophan. Gene order correlates with order of reactions in pathway. 5’ UTR (Leader) 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 Low High