XII. Gene Regulation

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 • ThelacOperon in E. coli • 1960 – Jacob and Monod proposed that this was an inducible system because the presence of the substrate INDUCES transcription. Promoter Repressor Gene Operator Repressor RNA Poly

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

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

18. XII. Gene Regulation • ThelacOperon in E. coli • Mutant analyses confirmed these results:

19. XII. Gene Regulation • ThelacOperon in E. coli • Mutant analyses confirmed these results:

20. XII. Gene Regulation • ThelacOperon in E. coli • Mutant analyses confirmed these results:

21. XII. Gene Regulation • ThelacOperon 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 • ThelacOperon 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 • ThelacOperon 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 • ThelacOperon 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 • ThelacOperon 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 • ThelacOperon 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 • ThelacOperon 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 lacoperon 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 • ThelacOperon in E. coli • B. The trpOperon in E. coli

30. XII. Gene Regulation • ThelacOperon in E. coli • B. The trpOperon 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 Secondary Regulation Actually, when trp is present,

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

35. B. The trp Operon in E. coli Secondary Regulation ACTUALLY, TRANSCRIPTION ALWAYS PROCEEDS A LITTLE BIT…UP TO THE REGION CALLED THE “ATTENUATOR”… Remember… in bacteria, there is no separation of transcription and translation. So, ribosomes attach to the RNA as it is being TRANSCRIBED…

36. B. The trp Operon in E. coli Secondary Regulation Two hairpin loops can form in the m-RNA: Sequence 3 can bind with either sequence 4 (terminating transcription) OR with sequence 2 (transcription on)

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

38. B. The trp Operon in E. coli Secondary Regulation Two hairpin loops can form in the m-RNA: 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…

39. B. The trp Operon in E. coli Secondary Regulation Two hairpin loops can form in the m-RNA: 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…

40. B. The trp Operon in E. coli Secondary Regulation Two hairpin loops can form in the m-RNA: Because translation occurs as soon as m-RNA is produced, ribosomes jump on and begin to read the strand… there are two trpcodons at the beginning of the sequence. If [trp] is LOW, then ribosome stalls at the trp codons; region 3 bind to 2, no termination loop forms, and transcription of the genes proceeds… So the enzymes that make tryptophan will be synthesized. Translation of the genes begins at start codons downstream…

41. XII. Gene Regulation • ThelacOperon in E. coli • B. The trpOperon in E. coli • C. Regulation in Eukaryotes

42. XII. Gene Regulation • ThelacOperon in E. coli • B. The trpOperon 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 just responding to environmental cues.

43. XII. Gene Regulation • ThelacOperon in E. coli • B. The trpOperon 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. • 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.

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

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

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

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

48. C. Regulation in Eukaryotes • Histone Regulation • Methylation • Promoters • 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

49. C. Regulation in Eukaryotes • Histone Regulation • Methylation • Promoters • 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.

50. C. Regulation in Eukaryotes • Histone Regulation • Methylation • Promoters • Enhancers/Silencers • These are DNA sequences upstream, downstream, or WITHIN the gene where Transcription Factors bind. Here are sites in the metallothionienIIA gene promoter region!! - What does having all these modifiers allow for?