1. Announcements 1. Reading Ch. 15: skim btm 425-426 2. Look over problems Ch. 15: 5, 6, 7.

2. Review of Last Lecture I. RNA processing in eukaryotes II. Translation of mRNA into protein - tRNA and ribosomes III. Three steps of translation IV. First evidence that proteins are important to heredity

3. Outline of Lecture 26 Regulating gene expression in prokaryotes I. Lactose metabolism - the lac operon, an inducible system II. Trp operon, a repressible system In E. coli, some proteins are found at 5-10 copies/cell; others are found at 100,00 copies. How does a cell regulate the levels or amounts of different proteins at different times - in response to the environment?

4. Operon: a series of gene coding regions (usually products with related functions) under the control of a single gene regulatory unit. Allows rapid response to changing environmental conditions. Background on gene organization lac operon 3 structural genes

5. Bacterial Strategy If glucose is present, –then use glucose as a carbon source. If glucose is not present, and if lactose is present, –then use lactose (indirectly) as carbon source. Levels of enzymes needed to use lactose as carbon source increase dramatically when lactose is present; enzymes are inducible and lactose is the inducer.

6. The  -galactosidase Enzyme lacZ gene-> lacY gene -> Permease (entry of lactose into cell) lacA -> transacetylase

7. lac operon, a polycistronic mRNA Could the cell make varying amounts of lacZ and lacA RNA?

8. Genes involved in Lactose Metabolism RepressorEnzymelactose ?- removes Regulatortransporter toxic Proteinbyproducts Normally binds to Operator when lactose is absent, but when lactose is present, comes off.

9. The Operon Model - components R L pol

10. R R Lac operon when no lactose is present

11. R R pol R R L L

12. R R

13. Learning Check pol R L Will transcription and translation of Z, Y,and A enzymes occur? What would happen if a wild-type copy of I was added?

14. glucoselactoseb-galactosidase +-- ++- --- -++ What happens when cell has both lactose and glucose? How does cell prevent breaking lactose down into glucose and galactose? Catabolite Activating Protein (CAP)

15. Catabolite Repression of lac Operon -/+ Glucose Therefore, for maximal transcription, repressor must be bound by repressor and CAP must bind CAP-binding site

16. Goal: efficiency, don’t waste energy converting lactose, when glucose available

17. Glucose Inhibits Formation of cAMP from ATP glucose

18. lac operon regulatory binding sites CAP site promoter operator lac coding Glu cAMP lactose lac rep + - - + - + CAP-cAMP lac rep - + - CAP-cAMP mRNA - + + RNA polymerase

19. 3-D structure of Repressor bound to Operator and CAP bound to Promoter Lewis et al. (1996) Science 271:1247 glucose absent lactose absent CAP Repressor Polymerase binds here DNA

20. II. Tryptophan Operon It makes sense for E. coli to synthesize the enzymes to make the amino acid tryptophan ONLY when: 1. The tryptophan concentration is low AND 2. The tryptophan-charged tRNA is low

21. Tryptophan Operon - Repressor Binds when tryptophan is present

22. mRNA leader sequence involved in Attenuation - the cast of characters

23. High Tryptophan- tRNA -->> Terminates Transcription Low Tryptophan- tRNA -->> Transcription Continues Translation Event controls Transcription Event !?!?