1. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Control of Gene Expression, Manipulating Genes Tobias Schoep

2. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Refresher: Translation

3. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Read the chapter 7 in your text book, Essential Cell Biology (3 rd Edition) Questions to schoep@engineering.ucsb.edu, Rm 3114

4. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Eukaryotes and prokaryotes have different mRNA transport for translation

5. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Eukaryotes and prokaryotes have different sequences upstream of ATG (start codon) Kozak sequence Is not ribosome binding site RBS is 5’ cap of mRNA Essential for translation initiation Shine Dalgarno sequence Is the ribosome binding site E.coli Human

6. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Eukaryotes and prokaryotes have different mRNA transport for translation

7. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 A molecule called transfer RNA (tRNA) recognizes the messenger RNA (mRNA) codon and adds the corresponding amino acid

8. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Amino acid specific synthetase enzymes “charge” the tRNA with the corresponding amino acid.

9. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Amino acid specific synthetase enzymes “charge” the tRNA with the corresponding amino acid.

10. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Ribosome de-codes (translates) mRNA into a new protein using charged tRNA The ribosome is a composed of a large and small subunit, proteins and structural RNA called ribosomal RNA (rRNA) Ribosome moves along the mRNA, captures complementary tRNA, hold these in position, and covalently links amino acids together. Aminoacyl-tRNA Peptidyl-tRNA Exit

11. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Ribosome produces protein in a 4 step process

12. LARGE SMALL

13. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 How does this process all start? Starts with binding of the initiator tRNA, which is always bound to Met, and binds START codon (AUG) Eukaryote: 5’ cap tells ribosome where to start searching for START single, spliced, mRNA transcribed for each protein Prokaryotes: ribosome recognizes ribosome binding sequence (RBS)– Shine dalgarno sequence ribosome can recognize multiple START sites in one mRNA molecule as long as RBS is present one mRNA protein can encode several different proteins

14. Eukaryote

15. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 How does this process all end? Ends with binding of the release factor binding to stop codon (UAG)

17. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Ribosome is relatively conserved between eukaryotes and prokaryotes Eukaryote ribosome adds about 2 aa / s, prokaryote ribosome adds about 20 aa / s Multiple ribosomes can be bound to one mRNA - polyribosomes Initiation, translation and termination

18. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11

19. Control of Gene Expression, Manipulating Genes Tobias Schoep

20. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Read the chapter 8 in your text book, Essential Cell Biology (3 rd Edition) Questions to schoep@engineering.ucsb.edu, Rm 3114

21. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Why is controlling gene expression important? All cell have the same DNA Eukaryotes: 25 000 genes – 5000 to 15000 expressed Prokaryotes: 4300 genes Some housekeeping genes are always produced How do we get different cells with different activities? Permanent and transient changes to gene expression such as 1.Cellular differentiation (permanent) 2.Cellular responses to stimuli (transient)

22. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Permanent changes to gene expression: Propagation of condensed chromatin structure Positive feed back loop (eukaryotes)

23. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Permanent changes to gene expression: DNA methylation (eukaryotes and prokaryotes) influences how regulatory proteins bind to DNA Pattern changed in fetal development affects cellular differentiation

24. Transient changes to gene expression: Changes to environmental stimuli Primary control is at the level of transcription ie. how much mRNA is produced Differences between eukaryotes and prokaryotes ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11

26. Transient changes to gene expression: Pokaryotes have operons: multiple genes produce one mRNA transcript and are regulated together. Eukaryotes generally have individual regulated genes but regulation is more complex ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11

27. 27 Repressor and Activators: Repressors turn off genes, activators turn them on Activator and repressor control of the Lac Operon in bacteria (prokaryote) Controls genes for using different carbon sources E.coli use glucose before lactose Enzymes for glucose use are made constantly Enzymes for lactose use are made at very low levels in the presence of glucose When glucose runs out and lactose is present Lac Operon is “turned on” Lac operon encodes genes for lactose transport (lacY) and conversion (lacZ) to glucose and galactose, and lacA

28. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 lactose active repressor (LacI) inactive repressor cAMP - ↓ glucose inactive activator active activator Components:

29. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 active repressor active activator Activator and Repressor binding sites:

30. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 ↓cAMP cAMP - ↓ glucose lactose active repressor active activator

31. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 ↑cAMP Lac operon based expression systems are commonly used to produce proteins

32. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Enhancers: Eukaryotes use activators and repressors as well. Activators bind to regions called enhancers Enhancers can bind to affect transcription from great distances In general: Activation depends on presence of multiple factors with act in combination Genes are often controlled by multiple sets of regulators ie. same gene can be expressed in response to different sets of signals However, a change in one transcription factor can cause profound changes in cell function

33. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11

34. Other mechanisms of regulating gene expression

35. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Other mechanisms of regulating gene expression Post-transcriptional controls operate after RNA polymerase has produced mRNA Conformational change in RNA can regulate gene expression

36. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 RNA switch affecting transcription (Riboswitch)

37. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Riboswitch Antisense RNA RNA switches affecting translation

38. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 RNA switches affecting RNA processing and degradation microRNA (miRNA) regulates up to 1/3 of protein coding genes RNA-induced silencing complex

39. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 RNA switches affecting RNA processing and degradation RNA interference (iRNA) detects foreign double stranded RNA (some viruses) Triggers cleavage to form small interfering RNAs (siRNA) Bind RISC and target foreign RNA for degradation

40. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 SUMMARY

41. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 What is the relevance of all this information for genetic and chemical engineers? Using expression systems from prokaryotes and eukaryotes we can produce proteins and other metabolites when and where we want them. Different systems can be used for prokaryotic cell: eg. ara/lac/trp expression systems eukaryotic cell: eg. siRNA to study gene function in mammalian cells animals: eg. Tet ON/OFF system to study gene function in animals

42. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Inducible gene expression system in animals Use of Tet ON/OFF system to study gene function in animals Turn on and off genes in animals Tetraycycline (antibiotic) responsive system Tetracycline approve human therapy

43. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Based on bacterial antibiotic resistance system Small amount of tetracycline turns on genes coding pumps to export tetracycline Components: tetR tetOtetA TetR PP tetR tetOtetA TetR PP tetracycline

44. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Tet (OFF) system Altered for use in eukaryotes Turn the TetR repressor into tetraycycline transactivator (tTA) Fused VP16 to C-terminus of TetR VP16 is from Herpes simplex virus and recruits RNA polymerase to site of promoter Seven copies of TetO upstream of promoter of gene of interest (GOI). Fusion of 7 operators is called the tetracycline response element (TRE)

45. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Tet (OFF) system In presence of tetracycline or doxycycline (tetracycline derivative), tTA (tetracycline transactivator) does not bind TetO in tetracycline response element (TRE), and there is reduced expression tetR P TREGOI P VP16 tTA activation TREGOI P tTA doxycycline

46. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Tet (ON) system Reverse system. Mutated TetR (rTetR) binds tetO in TRE in presence of tetracycline Modified TetR fused to VP16 is the tetraycycline responsive transactivator (rtTA)

47. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Tet (ON) system In presence of tetracycline or doxycycline (tetracycline derivative), rtTA (tetracycline responsive transactivator) does bind TetO in tetracycline reseponse element (TRE), and there is increased expression rtetR P TREGOI P VP16 rtTA activation TREGOI P tTA doxycycline

48. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 More advanced tet based systems Tet-On Advanced and Tet-On 3G: Increased sensitivity to doxycycline and lower basal expression. All very interesting, but what are the tet systems good for 1.Expression of reporter systems in animals such that localization of protein expression can be examined 2.Deletion of specific cells in animals such that cell function and regeneration can be examined 3.Construction of models that mimic human diseases 4.Regulatable models of tumorgenesis

49. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Tet (OFF): Regulatable models of tumorgenesis Study of hepatocelluar carcinoma (liver cancer) Examined role of MYC oncogene, gene with potential to cause cancer, at different stages of mouse development Beer et al. (2004) Developmental Context Determines Latency of MYC-Induce Tumors, Plos Biology, 2(11):1785-1798

50. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Used Tet (OFF) system to turn on MYC oncogene expression when doxycycline removed from mouse drinking water TetR P VP16 tTA Enhancer TREMYC P tTAactivation TREMYC P tTA doxycycline in drinking water Remove doxycycline from drinking water Liver specific basal myc ↑ myc

51. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Tet (ON): Study of transgenic cloned dogs Generation of transgenic dogs that conditionally express green fluorescent protein Transfer of DNA to an egg cell from which nuclear DNA had been removed Kim et al. (2011) Generation of Transgenic Dogs that Conditionally Express Green Fluorescent Protein, Genesis, 49:472–478 TREeGFP P rtTA activation doxycycline

52. ChE 170: Engineering Cell Biology – Control of gene expression, manipulating genes 11/03/11 Tet (ON): Addition of doxycycline causes induces eGFP production Kim et al. (2011) Generation of Transgenic Dogs that Conditionally Express Green Fluorescent Protein, Genesis, 49:472–478