1. XII. Gene Regulation

2. - Overview: All cells in an organism contain the same genetic information; the key to tissue specialization is gene regulation – reading some genes in some cells and other genes in other cells.

3. Also, organisms can respond to their environment at a genetic level, so there must be a way for the environment to stimulate or repress the action of certain genes.

4. And changes occur through time, creating developmental changes. We will look at how gene expression is regulated in these cases.

5. - Overview: -Some Terminology: - some enzymatic genes are only turned on if the substrate is present; this is an inducible system and the substrate is the inducer. Obviously, this is highly adaptive, as the cell saves energy by only producing the enzyme when it is needed.

6. - Overview: -Some Terminology: - some enzymatic genes are only turned on if the substrate is present; this is an inducible system and the substrate is the inducer. Obviously, this is highly adaptive, as the cell saves energy by only producing the enzyme when it is needed. - some enzymes are on all the time, and are only turned off if a compound (often the product of the metabolic process they are involved with) is present. This is a repressible system, and the compound is the repressor. This is also adaptive, and the cell saves on enzymes if the product is already present.

7. - Overview: -Some Terminology: - some enzymatic genes are only turned on if the substrate is present; this is an inducible system and the substrate is the inducer. Obviously, this is highly adaptive, as the cell saves energy by only producing the enzyme when it is needed. - some enzymes are on all the time, and are only turned off if a compound (often the product of the metabolic process they are involved with) is present. This is a repressible system, and the compound is the repressor. This is also adaptive, and the cell saves on enzymes if the product is already present. - Constitutive genes are on all the time.

8. XII. Gene Regulation A. The lac Operon in E. coli

9. XII. Gene Regulation A. The lac Operon in E. coli - When lactose is present, E. coli produce three enzymes involved in lactose metabolism. Lactose is broken into glucose and galactose, and galactose is modified into glucose, too. Glucose is then metabolized in aerobic respiration pathways to harvest energy (ATP). When lactose is absent, E. coli does not make these enzymes and saves energy and amino acids. How do they KNOW? : )

10. XII. Gene Regulation A. The lac Operon in E. coli As you remember, an “operon” was a region of genes that are regulated as a unit – it typically encodes > 1 protein involved in a particular metabolic pathway.

11. XII. Gene Regulation A. The lac Operon in E. coli As you remember, an “operon” was a region of genes that are regulated as a unit – it typically encodes > 1 protein involved in a particular metabolic pathway.

12. XII. Gene Regulation A. The lac Operon in E. coli Lac Y - permease – increases absorption of lactose

13. XII. Gene Regulation A. The lac Operon in E. coli Lac Y - permease – increases absorption of lactose Lac Z – B-galactosidase – cleaves lactose into glucose and galactose

14. XII. Gene Regulation A. The lac Operon in E. coli Lac Y - permease – increases absorption of lactose Lac Z – B-galactosidase – cleaves lactose into glucose and galactose Lac A – transacetylase – may code for enzymes that detoxify waste production of digestion.

15. XII. Gene Regulation A.The lac Operon in E. coli 1960 – Jacob and Monod proposed that this was an inducible system because the presence of the substrate INDUCES transcription. Repressor Gene RepressorRNA Poly Operator Promoter

16. XII. Gene Regulation A.The lac Operon in E. coli 1960 – Jacob and Monod proposed that this was an inducible system because the presence of the substrate INDUCES transcription. LACTOSE

17. XII. Gene Regulation A.The lac Operon in E. coli 1960 – Jacob and Monod proposed that this was an inducible system because the presence of the substrate INDUCES transcription. LACTOSE The binding of lactose changes the shape of the repressor (allosteric reaction) and it can’t bind to the operator.

18. XII. Gene Regulation A.The lac Operon in E. coli Mutant analyses confirmed these results:

19. XII. Gene Regulation A.The lac Operon in E. coli Mutant analyses confirmed these results:

20. XII. Gene Regulation A.The lac Operon in E. coli Mutant analyses confirmed these results:

21. XII. Gene Regulation A.The lac Operon in E. coli Mutant analyses confirmed these results: Curiously, there are only about 10 repressor molecules in each cell and they were not actually isolated and identified for 6 years (Gilbert).

22. XII. Gene Regulation A.The lac Operon in E. coli But it is even more complicated… if glucose AND lactose are present, the operon is OFF. This is adaptive, because it’s glucose the cell needs. If glucose is present, there is no need to break lactose down to get it. BUT HOW?

23. XII. Gene Regulation A.The lac Operon in E. coli But it is even more complicated… if glucose AND lactose are present, the operon is OFF. This is adaptive, because it’s glucose the cell needs. If glucose is present, there is no need to break lactose down to get it. BUT HOW? This involves a repressible pathway.

24. XII. Gene Regulation A.The lac Operon in E. coli Within the promoter, there is a binding site for Catabolic Activating Protein – basically a “transcription factor”. CAP needs to bind in order for the RNA Polymerase to bind. Cyclic-AMP activates CAP, causing an allosteric reaction so it can bind the promoter., lactose present

25. XII. Gene Regulation A.The lac Operon in E. coli Within the promoter, there is a binding site for Catabolic Activating Protein – basically a “transcription factor”. CAP needs to bind in order for the RNA Polymerase to bind. Cyclic-AMP activates CAP, causing an allosteric reaction so it can bind the promoter. So, the binding of CAP stimulates transcription., lactose present

26. XII. Gene Regulation A.The lac Operon in E. coli When Glucose is present, the concentration of c-AMP declines, it does not bind to CAP, and CAP does not bind to the Promoter; so the RNA Poly does not bind either and the genes are off., lactose present

27. CAP REPRESSOR

28. XII. Gene Regulation A.The lac Operon in E. coli When Glucose is present, the concentration of c-AMP declines, it does not bind to CAP, and CAP does not bind to the Promoter; so the RNA Poly does not bind either and the genes are off. So, the lac operon is regulated first by the presence/absence of glucose; the needed nutrient…and then by the presence of lactose, which could be metabolized to produce glucose if necessary.

29. XII. Gene Regulation A.The lac Operon in E. coli B. The trp Operon in E. coli

30. XII. Gene Regulation A.The lac Operon in E. coli B. The trp Operon in E. coli Tryptophan is an amino acid that can be synthesized by tryptophan synthetase. This gene and its partners are only ON if tryptophan is absent. The presence of tryptophan represses the production of these enzymes (repressible system).

31. B. The trp Operon in E. coli If trp is absent, the repressor can’t bind to the operator… transcription proceeds..

32. B. The trp Operon in E. coli If trp is present, it binds to the repressor, changing the repressor’s shape so that it can now bind to the operator and inhibit RNA poly binding.

33. B. The trp Operon in E. coli Actually, when trp is present, Secondary Regulation

34. B. The trp Operon in E. coli ACTUALLY, TRANSCRIPTION ALWAYS PROCEEDS A LITTLE BIT…UP TO THE REGION CALLED THE “ATTENUATOR”… Secondary Regulation

35. B. The trp Operon in E. coli ACTUALLY, TRANSCRIPTION ALWAYS PROCEEDS A LITTLE BIT…UP TO THE REGION CALLED THE “ATTENUATOR”… Secondary Regulation

36. B. The trp Operon in E. coli Two hairpin loops can form in the m- RNA; the 3-4 loop causes termination of transcription. Secondary Regulation

37. B. The trp Operon in E. coli Two hairpin loops can form in the m- RNA; the 3-4 loop causes termination of transcription. Because translation occurs as soon as m-RNA is produced, ribosomes jump on and begin to read the strand… there are two trp codons at the beginning of the sequence. Secondary Regulation

38. B. The trp Operon in E. coli Two hairpin loops can form in the m- RNA; the 3-4 loop causes termination of transcription. Because translation occurs as soon as m-RNA is produced, ribosomes jump on and begin to read the strand… there are two trp codons at the beginning of the sequence. If trp is present, the ribosome zooms along (incorporating trp) and it occupies the 2 region… region 3 is free to bind with 4 and the termination loop forms… Secondary Regulation

39. B. The trp Operon in E. coli Two hairpin loops can form in the m- RNA; the 3-4 loop causes termination of transcription. Because translation occurs as soon as m-RNA is produced, ribosomes jump on and begin to read the strand… there are two trp codons at the beginning of the sequence. If trp is present, the ribosome zooms along (incorporating trp) and it occupies the 2 region… region 3 is free to bind with 4 and the termination loop forms… If low trp, then ribosome stalls; region 3 bind to 2, no termination loop forms, and transcription of the genes proceeds… Translation of the genes begins at start codons downstream… Secondary Regulation

40. XII. Gene Regulation A.The lac Operon in E. coli B. The trp Operon in E. coli C. Regulation in Eukaryotes

41. XII. Gene Regulation A.The lac Operon in E. coli B. The trp Operon in E. coli C. Regulation in Eukaryotes - higher levels of packaging, intron-exon structure, and the need for tissue specialization makes regulation in eukaryotes far more complex than responding to environmental cues.

42. XII. Gene Regulation A.The lac Operon in E. coli B. The trp Operon in E. coli C. Regulation in Eukaryotes - higher levels of packaging, intron-exon structure, and the need for tissue specialization makes regulation in eukaryotes far more complex that responding to environmental cues. 1.Histone Regulation - Core DNA, bound to histones, is OFF. Only “linker DNA”, between histones, is even accessible to RNA polymerases. So, binding DNA to histones is the first way to shut it off.

43. C. Regulation in Eukaryotes 1.Histone Regulation - Three ways to reveal DNA “chromatin remodeling”

44. C. Regulation in Eukaryotes 1.Histone Regulation - Three ways to reveal DNA “chromatin remodeling” 2.Methylation - highly repetitive sequences - imprinted genes - Barr bodies

45. C. Regulation in Eukaryotes 1.Histone Regulation - Three ways to reveal DNA “chromatin remodeling” 2.Methylation - highly repetitive sequences - imprinted genes - Barr bodies Some proteins bind to the methylated cytosines, and may either recruit repressors or interrupt transcription factor binding.

46. C. Regulation in Eukaryotes 1.Histone Regulation 2.Methylation 3.Promoters - Several consensus sequences (TATA, CAAT, GGGCGG) appear in combination in nearly all promoters and are required for basal levels of transcription

47. C. Regulation in Eukaryotes 1.Histone Regulation 2.Methylation 3.Promoters 4.Enhancers/Silencers Cis-acting elements on the same chromosome, which regulate a neighboring gene. They are somewhat like operators, in that they are binding sites for transcription factors that can “up” or “down” regulate transcription. However, they function ANYWHERE near the gene: before, within, or after

48. C. Regulation in Eukaryotes 1.Histone Regulation 2.Methylation 3.Promoters 4.Enhancers/Silencers Cis-acting elements on the same chromosome, which regulate a neighboring gene. They are somewhat like operators, in that they are binding sites for transcription factors that can “up” or “down” regulate transcription. However, they function ANYWHERE near the gene: before, within, or after They are not gene specific – they will enhance their neighbor Silencers tend to reduce binding of the polymerase to the promoter.

49. C. Regulation in Eukaryotes 1.Histone Regulation 2.Methylation 3.Promoters 4.Enhancers/Silencers These are the transcription factors that bind to enhancer and silencer regions of the human metallothionien IIA gene promoter region!! - What does having all these modifiers allow for?

50. Different proteins can silence or enhance DNA Polymerase II binding. This may involve the formation of a pre-initiation complex” of proteins that allow the Poly II to bind, and can even involve sequences far from the promoter that loop – and influence RNA Poly II activity.

51. C. Regulation in Eukaryotes 4.Enhancers/Silencers 5.Transcription Factors - These are the proteins that bind to DNA and influence transcription. They have “binding domains” that bind DNA in particular ways.

52. C. Regulation in Eukaryotes 4.Enhancers/Silencers 5.Transcription Factors - These are the proteins that bind to DNA and influence transcription. They have “binding domains” that bind DNA in particular ways. HTH = “helix-turn-helix” One class of important HTH TF’s contain specific sequences of AA’s called a homeodomain. This is encoded by a 180 bp region in it’s gene called a homeobox. These homeotic genes/proteins are conserved across all eukaryotes and are critical to basic animal development.

53. C. Regulation in Eukaryotes 4.Enhancers/Silencers 5.Transcription Factors - These are the proteins that bind to DNA and influence transcription. They have “binding domains” that bind DNA in particular ways. “Zinc-Finger”: Zinc binds to two cysteine and two histidine AA’s. The sequence between forms A loop or “finger”, and the specific AA sequence Binds specific DNA sequences…

54. C. Regulation in Eukaryotes 4.Enhancers/Silencers 5.Transcription Factors We didn’t really know what they did in vivo. Biochemists have linked other proteins to them, however, making Zinc- finger nucleases that cut DNA at specific sequences.

56. C. Regulation in Eukaryotes 4.Enhancers/Silencers 5.Transcription Factors But Feb 18, 2015, Najafabadi et al. found this: - “ Cys2-His2 zinc finger (C2H2-ZF) proteins represent the largest class of putative human transcription factors” (> 700 proteins) - “C2H2-ZF proteins recognize more motifs than all other human transcription factors combined.” (Highly variable, tough to study) - “C2H2-ZF proteins bind specific endogenous retroelements (EREs), ranging from currently active to ancient families. The majority of C2H2-ZF proteins, also show widespread binding to regulatory regions, indicating that the human genome contains an extensive and largely unstudied adaptive C2H2-ZF regulatory network that targets a diverse range of genes and pathways.” - They stabilized Endogenous Retroviral Elements, and evolved to regulate other genes, as well. Najafabadi et al. 2015 Science Daily READ THIS

57. C. Regulation in Eukaryotes 4.Enhancers/Silencers 5.Transcription Factors - These are the proteins that bind to DNA and influence transcription. They have “binding domains” that bind DNA in particular ways. bZIP=“basic leucine zipper”: leucine AA’s in Different chains dimerize and the leucines “zip” The other alpha-helices bind specific DNA sequences

58. C. Regulation in Eukaryotes 4.Enhancers/Silencers 5.Transcription Factors - These are the proteins that bind to DNA and influence transcription. They have “binding domains” that bind DNA in particular ways. - Then, the TF’s have other binding sites for Proteins (like basal transcription factors) or Other chemicals (like hormones)

59. C. Regulation in Eukaryotes 5.Transcription Factors 6.Alternate Splicing Pathways - Many proteins can be made from the same gene, by splicing the m-RNA differently. Humans have 20-30K genes, but several 100,000 proteins! A calcium regulator in the thyroid A hormone made in the brain

60. C. Regulation in Eukaryotes 6.Alternate Splicing Pathways 7. Controlling m-RNA stability Existing tubulin units interact with a new tubulin strand and translation stalls, releasing RNAse that cleave the m-RNA. So tubulin is only made when free tubulin units are not present.

61. C. Regulation in Eukaryotes 7.Controlling m-RNA stability 8.RNA Silencing - Short pieces of RNA can bind to DNA in the nucleus or m-RNA in the cytoplasm and regulate gene expression.

62. C. Regulation in Eukaryotes 7.Controlling m-RNA stability 8.RNA Silencing/Interference - si-RNA (small interfering RNA): Viral or retrotransopon origin - mi-RNA (micro-RNA): Produced by intronic, sequences, or different genes in genome. Have stem-loop structure.

63. C. Regulation in Eukaryotes 7.Controlling m-RNA stability 8.RNA Silencing/Interference THEY BOTH are attacked by DICER protein, which cuts them into short ds-RNA molecules. These complex with RNA-induced Silencing Complex proteins (RISC) that denature the RNA and degrade the sense strand. What is left is a strand that is complementary to a specific m-RNA molecule.

64. C. Regulation in Eukaryotes 7.Controlling m-RNA stability 8.RNA Silencing/Interference If the ss-RNA is exactly complementary to a m-RNA, RISC cuts the m-RNA into fragments (turning protein synthesis OFF). Or, if not exactly complementary, then the RISC complex stays attached, interrupting ribosome binding and translation.

65. C. Regulation in Eukaryotes 7.Controlling m-RNA stability 8.RNA Silencing/Interference OR! The ds-RNA gets complexed with RNA-induced initiation of transcription silencing complex (RITS). These denature the RNA, creating ss- RNA that binds to DNA promoters or large regions of DNA. This binding attracts chromatin remodeling proteins that methylates the histones, causing it to coil into herochromatin (Turning Genes OFF).

66. The process of Gene Activity – in terms of a Gene Making a Functional Protein – can be regulated at every step of the process, from: Gene availability and chromatin structure Transcription Transcript Processing Translation Post-translational Modification Variation in patterns of regulation lead to differences in expression between cells, and cell specialization.