Prokaryotic Gene Regulation

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

2. Types of Control

3. 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)

4. Types of Operons

5. Regulation of the Lac Operon crp O Pcrp Pi P Lac Z Lac Y Lac A I Structural Genes

6. O Pcrp crp Pi P Lac Z Lac Y I Lac A Pol 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. Transcription from the Lac Operon

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

8. O Pcrp crp Pi P Lac Z Lac Y Lac A I Pol Transcription I mRNA crp mRNA Z, Y, A mRNA Translation + Lactose Active repressor Inactive CAP protein 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, High glucose

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

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

11. 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

12. Summary of Lac Operon Regulation Off Off On at low frequency On at high frequency

13. Mutations of the Lac Operon O Pcrp Pi CRP P Lac Z Lac Y Lac A I Functional genes: I+ P+ O+ Z+ Y+ A+ 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).

14. Mutations of the Lac Operon O Pcrp Pi CRP P Lac Z Lac Y Lac A I Functional genes: I+ P+ O+ Z+ Y+ A+

15. Mutations of the Lac Operon O Pcrp Pi CRP P Lac Z Lac Y Lac A I 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. B-Galactosidase Permease Lac Operon Mutations, Page 3-24 No lactose Lactose No lactose Lactose -- + -- + + + + + -- -- -- -- + + + + -- + -- + + + -- + -- + + + -- -- -- -- -- -- -- --

17. Arabinose Operon

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

19. Summary of Ara Operon Regulation OffC protein bound to O and I,Inhibiting transcription OffC protein bound to O and I On at low frequencyC protein + arabinose bound to I, enhancing transcription On at high frequencyC protein + arabinose bound to I and cAMP + CAP boundto I, enhancing transcription

20. trpA Tryptophan Operon

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

22. 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

23. Termination signal due to hairpin formed by 3+4 pairing followed by string of uracils No terminationsignal formed Alternative RNA Structures from 5’ UTR Formation of termination signal depends on level of tryptophan carried by tRNA in the cell.

24. AttenuationPremature 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 continuestoward trp E, D, C. B, A

26. Summary of Trp Operon Regulation OnTrp repressor inactive Lack of attenuation leads to high rate of mRNA production OffTryptophan + repressor = Active repressor Reduction of mRNA production by attenuation