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

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

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

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

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

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

11. How do genetic switches work?

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

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

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. mRNA 5' DNA mRNA Protein Allolactose (inducer) Inactive repressor lacl lacz lacYlacA RNA polymerase PermeaseTransacetylase  -Galactosidase 5 3 mRNA 5 The Lac operon Figure 18.22b What will happen if there is a deletion of the: + lactose?- lactose? operator? lac repressor gene? CAP binding site?

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? OgalEOgalTgalK Gal repressor protein (galR) EpimeraseTransferaseKinase P Don’t memorize these names- just the general concept. CAP Galactose

20. Gene Regulation in Prokaryotes and Eukarykotes 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

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

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

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

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