1. Prokaryotic Gene Regulation Coordinate regulation of genes involved in similar functions

2. Types of Control Negative Control Product of regulatory gene inhibits transcription Positive Control Product of regulatory gene enhances transcription

3. Operon Unit of coordinate gene expressionUnit of coordinate gene expression Includes structural genes and their adjacent regulatory elementsIncludes structural genes and their adjacent regulatory elements We will considerWe will consider –Lac operon (inducible) –Ara operon (inducible) –Trp operon (repressible)

4. Types of Operons Inducible Initial condition: OFF Inducer switches operon ON Repressible Initial condition: ON Repressor switches operon OFF

5. Lac ZLac YLac AP O PiPi I P crp crp Regulation of the Lac Operon DNA Function Protein Function P crp Promoter for crp gene crp Gene for CAP protein Positive regulator PiPiPiPi Promoter for I gene I Gene for Lac Repressor Negative regulator P Promoter for Structural Genes OOperator Lac Z Gene for B-galactosidase Cleaves lactose Lac Y Gene for Permease Lactose transport Lac A Gene for Acetylase Unknown Structural Genes

6. Lac ZLac Y Lac A P O PiPi I P crp crp Transcription from the Lac Operon 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-galactosidaseTranslationPermeaseAcetylase

7. Lac ZLac YLac AP O PiPi I P crp crp Regulation of the Lac Operon: Low lactose, High glucose High glucose Transcription from P crp and P i is constitutive: always expressed in an unregulated fashion. Active repressor binds to operator and prevents RNA polymerase from reaching structural genes. Activerepressor crp mRNA I mRNA Inactive CAP protein No mRNA produced No Z, Y, A proteins produced Pol

8. Regulation of the Lac Operon: High lactose, High glucose High glucose Lac ZLac YLac AP O PiPi I P crp crp Activerepressor crp mRNA I mRNA Inactive CAP protein Lactose Lactose (inducer) binds to the repressor and inactivates it. RNA polymerase transcribes Lac Z, Y and A at low frequency. +Inactiverepressor Pol Z, Y, A mRNA Transcription TranslationB-galactosidasePermeaseAcetylase

9. Regulation of the Lac Operon: High lactose, Low glucose Low glucoseTranslationB-galactosidasePermeaseAcetylase Lac ZLac YLac AP O PiPi I P crp crp Activerepressor crp mRNA I mRNA Inactive CAP protein Lactose + Inactiverepressor + 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 Pol Z, Y, A mRNA Transcription

10. Regulation of the Lac Operon: Low lactose, Low glucose Low glucose Activerepressor Lac ZLac YLac AP O PiPi I P crp crp crp mRNA I mRNA Inactive CAP protein cAMP + 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. No mRNA produced No Z, Y, A proteins produced Pol

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

12. Summary of Lac Operon Regulation Level of Lactose Level of Glucose Lac Operon LowHigh LowLow HighHigh HighLow Off Off On at low frequency On at high frequency

13. Lac ZLac YLac AP O PiPi I P crp CRP Mutations of the Lac Operon I+ I+ I+ I+ Functional Repressor Trans-acting I- I- I- I- Non-functional Repressor Trans-acting Is Is Is IsSuperrepressor (cannot bind lactose) Trans-acting I s > I + > I - Functional genes: I + P + O + Z + Y + A + The diffusible product of the I + or I S allele can associate with an operator on the same piece of DNA (cis) or on a separate piece of DNA (trans).

14. Lac ZLac YLac AP O PiPi I P crp CRP Mutations of the Lac Operon P+P+P+P+ Functional Promoter Cis-acting P-P-P-P- Non-functional Promoter Cis-acting O+O+O+O+ Functional Operator Cis-acting OcOcOcOc Non-functional Operator (Operator Constitutive) Cis-acting O-O-O-O- Non-functional Operator (Operator region deleted) Cis-acting Functional genes: I + P + O + Z + Y + A +

15. Lac ZLac YLac AP O PiPi I P crp CRP Mutations of the Lac Operon Z+Z+Z+Z+ Functional B-galactosidase Z-Z-Z-Z- Non-functional gene for B-galactosidase Y+Y+Y+Y+ Functional Permease Y-Y-Y-Y- Non-functional gene for Permease A+A+A+A+ Functional Acetylase A-A-A-A- Non-functional gene for Acetylase Functional genes: I + P + O + Z + Y + A + A mutation in one structural gene does not affect the production of proteins from the other structural genes.

16. Lac Operon Mutations, Page 3-24 I+P+O+Z+Y+I+P+O+Z+Y+I+P+O+Z+Y+I+P+O+Z+Y+ I-P+O+Z+Y+I-P+O+Z+Y+I-P+O+Z+Y+I-P+O+Z+Y+ I+P-O+Z+Y+I+P-O+Z+Y+I+P-O+Z+Y+I+P-O+Z+Y+ I+P+OcZ+Y+I+P+OcZ+Y+I+P+OcZ+Y+I+P+OcZ+Y+ I + P + O + Z - Y + / I - P + O + Z + Y + I + P + O c Z + Y - / I + P + O + Z - Y + I + P + O c Z - Y + / I - P + O + Z + Y - I s P + O + Z + Y - / I - P + O + Z - Y + I + P - O c Z + Y + / I + P + O + Z - Y - No lactose LactoseLactose B-GalactosidasePermease -- + -- + + + + + -- -- -- -- + + + + -- + -- + + + -- + + + -- + -- + + + -- + + + -- -- -- --

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

18. C Protein Exerts Positive and Negative Control of the Ara Operon Arabinose present Arabinose absent

19. Summary of Ara Operon Regulation Level of Arabinose Level of Glucose Ara Operon LowHigh LowLow HighHigh HighLow Off C protein bound to O and I, Inhibiting transcription Off C protein bound to O and I On at low frequency C protein + arabinose bound to I, enhancing transcription On at high frequency C protein + arabinose bound to I and cAMP + CAP bound to I, enhancing transcription

20. DNA Function RNA/Protein Function Trp R Gene for repressor Binds to operator to inhibit transcription PPromoter OOperator 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 Tryptophan Operon trpA

21. Control of Trp Operon Transcription Trp Repressor is Inactive  Initial State: ON Trp binding activates Repressor  Final State: OFF tryptophan

22. Features of the 5’ UTR Contains complementary sequences that can form hairpin structures when transcribed into RNAContains 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 structureCodes 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 productCodes for several Trp codons as part of an unstable protein product

23. 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.

24. Attenuation Premature Termination of Transcription Ribosome translates trp codons, preventing 2+3 pairing 3+4 pairing forms terminator

25. Antitermination Ribosome stalls at trp codons, allowing 2+3 pairing Transcription continues toward trp E, D, C. B, A

26. Summary of Trp Operon Regulation Level of Tryptophan Trp Operon Low High On Trp repressor inactive Lack of attenuation leads to high rate of mRNA production Off Tryptophan + repressor = Active repressor Reduction of mRNA production by attenuation