32 Gene regulation, continued

1. 32 Gene regulation, continued

2. Lecture Outline 11/21/05 • Review the operon concept • Repressible operons (e.g. trp) • Inducible operons (e.g. lac) • Positive regulation of lac (CAP) • Practice applying the operon concept to predict: • the phenotypes of mutants • The characteristics of other operons • Gene regulation in prokaryotes vs eukaryotes

3. trp operon Promoter RNA polymerase Polypeptides that make up enzymes for tryptophan synthesis The trp operon: Tryptophan absent -> repressor inactive -> transcription Regulatory gene Genes of operon trpR trpD trpC trpB trpE trpA DNA Operator mRNA 3 5 mRNA 5 C E D B A One long mRNA codes several polypeptides, each with its own start and stop codon Protein Figure 18.21a The “operator” is a particular sequence of bases where the repressor can bind

4. Active repressor can bind to operator and block transcription Trpoperon DNA No RNA made mRNA Protein Active repressor Tryptophan (corepressor) Tryptophan present -> repressor active -> operon “off”. Figure 18.21b

5. Lac operonInducible operons are normally off When lactose is present, repressor can no longer bind DNA. Transcription occurs

6. Positive vs Negative Gene Regulation • Both the trp and lac operons involve negative control of genes • because the operons are switched off by the active form of the repressor protein • Some operons are also subject to positive control • An activator protein is required to start transcription. • E.g. catabolite activator protein (CAP)

7. Positive Gene Regulation- CAP • In E. coli, glucose is always the preferred food source • When glucose is scarce, the lac operon is activated by the binding of CAP Promoter DNA lacl lacZ Operator Active form of CAP helps RNA polymerase bind to promoter, so transcription can start Figure 18.23a ActiveCAP cAMP Inactive lac repressor InactiveCAP

8. First messenger (signal molecule such as epinephrine) Adenylyl cyclase G protein GTP G-protein-linked receptor ATP cAMP Protein kinase A Cellular responses • Enzyme adenylylcyclase You’ve seen cAMP used in other signaling pathways

9. When glucose is abundant, • cAMP is used up • CAP detaches from the lac operon, • prevents RNA polymerase from binding to the promoter Promoter DNA lacl lacZ Operator RNA polymerase can’t bind InactiveCAP Inactive lac repressor Figure 18.23b

10. Glucose transporter complex also activates adenylate cyclase If it is busy phosphorylating glucose, it cannot activate adenylate cyclase, so level of cAMP falls

11. How do genetic switches work?

12. DNA binding proteins can be either repressors or activators, depending on how they intereact with RNA polymerase Activator This configuration helps RNA polymerase bind Repressor This configuration blocks RNA polymerase

14. Dual control of the lac operon Glucose must be absent Lactose must be present + glucose + lactose off, because CAP not bound + glucose - lactose off, because repressor active and CAP not bound - glucose - lactose off, because repressor active Operon active - glucose + lactose

15. X-ray structure of CAP-cAMP bound to DNA Many Operons use CAP lac, gal, mal, ara, etc. CAP binds to RNA polymerase

16. The Lac operon DNA lacl lacz lacY lacA RNApolymerase What will happen if there is a deletion of the: + lactose? - lactose? • operator? • lac repressor gene? • CAP binding site? 3 mRNA 5 mRNA mRNA 5' 5 -Galactosidase Permease Transacetylase Protein Inactiverepressor Allolactose(inducer) Figure 18.22b

17. Arabinose is another sugar that E. coli can metabolize • Will those genes be repressible or inducible? • How might it be regulated? Arabinose can bind to the repressor

18. Arginine is an essential amino acid. • Will that pathway be repressible or inducible? • How might argenine synthesis be regulated?

19. Galactose is yet another sugar that E. coli can metabolize. • Will those genes be repressible or inducible? • How might gal be regulated? CAP P O galE O galT galK Epimerase Transferase Kinase Don’t memorize these names- just the general concept. Galactose Gal repressor protein (galR)

20. Prokaryotes Operons 27% of E. coli genes (Housekeeping genes not in operons) simultaneous transcription and translation Eukaryotes No operons, but they still need to coordinate regulation More kinds of control elements RNA processing Chromatin remodeling Histones must be modified to loosen DNA Gene Regulation in Prokaryotes and Eukarykotes

21. Signal NUCLEUS Chromatin Chromatin modification: Gene available for transcription DNA Gene Transcription Exon RNA Primary transcript Intron RNA processing Tail mRNA in nucleus Cap Transport to cytoplasm CYTOPLASM mRNA in cytoplasm Degradation of mRNA Translation Polypetide Cleavage Chemical modification Transport to cellular destination Active protein Degradation of protein Degraded protein Figure 19.3

22. Protein scaffold Loops 30 nm 700 nm Scaffold 300 nm Nucleosome (c) Looped domains (300-nm fiber) (b) 30-nm fiber 1,400 nm (d) Metaphase chromosome DNA Packing Figure 19.2

23. Histone Modification Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation Histone tails DNA double helix Amino acids available for chemical modification Figure 19.4a

24. Acetylated histones Unacetylated histones Figure 19.4 b Histone acetylation loosens DNA to allow transcription