Warm-up

1. Warm-up • Pick up a study questionnaire and a reading guide packet (it’s all of them for this unit) • Reading guide – due on the test day • Study questionnaire complete in the next few minutes • Be as accurate as you can • Don’t put your name • When you are done, look over questions 1-17 in the reading guide (we are covering those in class) • This unit – lab heavy, we will have quizzes on chapters 18-20

2. Our Drive For 5 is just beginning!!!

3. Today • Review • Allosteric regulation • Protein structure • Transcription • New stuff • Look at the structure of the operon (found in bacteria) • Understand postive vs. negative gene regulation • Understand repressible vs. inducible regulation • Understand lac vs. trp operon regulation

4. Control of Prokaryotic (Bacterial) Genes

5. #1 - Why regulate genes? • Maintain homeostasis – gene expression is affected by changes in the external environment • Multicellular organisms – have specialized cells • All cells have the full complement of genes, but not all genes are needed in each cell (some are turned off)

6. Bacterial metabolism • Bacteria need to respond quickly to changes in their environment • if they have enough of a product, need to stop production • why? waste of energy to produce more • how? stop production of enzymes for synthesis • if they find new food/energy source, need to utilize it quickly • why? metabolism, growth, reproduction • how? start production of enzymes for digestion • Cost effective – natural selection favors cells that can do this well STOP GO

7. Enzyme Review

8. Allosteric Regulation

9. - - = inhibition # 3 - Remember Regulating Metabolism? • Feedback inhibition • product acts as an allosteric inhibitor of 1st enzyme in tryptophan pathway • but this is wasteful production of enzymes Oh, I remember thisfrom our Metabolism Unit!

10. - - - = inhibition #2 - Different way to Regulate Metabolism • Gene regulation • instead of blocking enzyme function, block transcription of genes for all enzymes in tryptophan pathway • saves energy by not wasting it on unnecessary protein synthesis Now, that’s a good idea from a lowly bacterium!

11. Overview - Gene regulation in bacteria • Cells vary amount of specific enzymes by regulating gene transcription • turn genes on or turn genes off • turn genes OFF exampleif bacterium has enough tryptophan then it doesn’t need to make enzymes used to build tryptophan • turn genes ON exampleif bacterium encounters new sugar (energy source), like lactose, then it needs to start making enzymes used to digest lactose STOP GO

12. (#’s 4,5,6,7) Operon Structure • Operon • genes grouped together with related functions • example: all enzymes in a metabolic pathway • promoter = RNA polymerase binding site • single promoter controls transcription of all genes in operon • transcribed as one unit & a single mRNA is made • operator = DNA binding site of repressor protein

13. # 8 So how can these genes be turned off? • Repressor protein • binds to DNA at operator site • blocking RNA polymerase (# 13) • blocks transcription (# 13) • Regulatory gene – codes for a regulatory protein, at different site than the gene it is regulating

14. RNA polymerase RNA polymerase repressor repressor enzyme1 1 enzyme2 2 enzyme3 3 enzyme4 4 promoter = repressor protein operator Operon model Operon: operator, promoter & genes they control serve as a model for gene regulation gene1 gene2 gene3 gene4 TATA DNA mRNA Repressor protein turns off gene by blocking RNA polymerase binding site.

15. trp RNA polymerase RNA polymerase repressor repressor repressor enzyme1 1 enzyme2 2 enzyme3 3 enzyme4 4 promoter repressor protein operator tryptophan trp trp trp trp trp trp trp trp trp tryptophan – repressor protein complex #10 - Repressible operon: tryptophan Synthesis pathway model When excess tryptophan is present, it binds to tryp repressor protein & triggers repressor to bind to DNA • blocks (represses) transcription • Usually on gene1 gene2 gene3 gene4 TATA DNA mRNA trp conformational change in repressor protein! trp

16. Tryptophan operon What happens when tryptophan is present? Don’t need to make tryptophan-building enzymes Tryptophan is allosteric regulator of repressor protein

17. RNA polymerase RNA polymerase repressor repressor repressor enzyme1 1 enzyme2 2 enzyme3 3 enzyme4 4 promoter repressor protein operator lactose lac lac lac lac lac lac lac lactose – repressor protein complex Inducible operon: lactose Digestive pathway model When lactose is present, binds to lac repressor protein & triggers repressor to release DNA • induces transcription • Usually off lac gene1 gene2 gene3 gene4 TATA DNA mRNA lac conformational change in repressor protein! lac

18. Lactose operon What happens when lactose is present? Need to make lactose-digesting enzymes Lactose is allosteric regulator of repressor protein

19. 1961 | 1965 Jacob & Monod: lac Operon • Francois Jacob & Jacques Monod • first to describe operon system • coined the phrase “operon” Jacques Monod Francois Jacob

20. # 17 - Operon summary • Repressible operon • Usually on • usually functions in anabolic pathways • synthesizing end products • when end product is present in excess, gene is turned off and cell allocates resources to other uses • Inducible operon • Usually off • usually functions in catabolic pathways, • digesting nutrients to simpler molecules • produce enzymes only when nutrient is available • cell avoids making proteins that have nothing to do, cell allocates resources to other uses

21. Lac operon • http://vcell.ndsu.edu/animations/lacOperon/index.htm

22. That’s not the whole story • So far, we have talked about negative regulation – repressor protein binds to the operator, turns off transcription • There is also positive regulation – activator binds to the promoter, turns transcription up (amplifies expression) • There are other nutrients bacteria can use besides lactose • Bacteria prefer glucose over lactose • Remember, glucose is needed for cell respiration to make ATP • When ATP is used up, it turns to ADP and even AMP

23. Cyclic AMP – when ATP levels are low

24. Fig. 18-5 Promoter Operator DNA lacI lacZ RNA polymerase binds and transcribes CAP-binding site Active CAP cAMP Inactive lac repressor Inactive CAP CAP Regulation Allolactose (a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized Promoter Operator DNA lacI lacZ CAP-binding site RNA polymerase less likely to bind Inactive CAP Inactive lac repressor (b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized

25. CAP,cAMP, and Glucose • As glucose supply increases, cAMP decreases, CAP is inactive, lac expression is slow/low • As glucose supply decreases, cAMP increases, CAP is active, lac expression is fast/high

26. Why is it regulated 2 ways? • Gives the bacteria choice • If a bacteria is given both glucose and lactose, it chooses glucose • Lactose causes the lac operon to be transcribed, but glucose (low cAMP) slows that transcription way down

27. Review • Negative Regulation – a repressor protein binds to the promoter and turns transcription off • Lac – inducer (repressor unbinds) • Trp – corepressor (repressor binds) • Positive Regulation – an activator binds to the promoter and turns transcription on • Lac – CAP is an activator • The lac operon is regulated in 2 ways • Overall themes – homeostasis and energy/nutrient conservation

28. Rest of Today • Complete the operon worksheet • Create a playdoh lac operon • Add the following to the list • activator (CAP) • cAMP • Check it with me – be ready to explain

29. Control of Eukaryotic Genes (we’re covering #’s18-36 today) PICK UP THE SHEETS AT THE FRONT

30. Calendar is almost done • Quiz – Ch. 18 is on Friday • Prokaryotic and eukaryotic regulation • Operon worksheet answers

31. Yesterday • Review • Allosteric regulation • Protein structure • Transcription • New stuff • Look at the structure of the operon (found in bacteria) • Understand postive vs. negative gene regulation • Understand repressible vs. inducible regulation • Understand lac vs. trp operon regulation

32. Today • Review • Prokaryotic vs Eukaryotic • New Stuff • Eukaryotic Regulation

33. Prokaryotic vs eukaryotic • Genomes • Protein synthesis • Cell respiration • ETC.

34. The BIG Questions… • How are genes turned on & off in eukaryotes? • How do cells with the same genes differentiate to perform completely different, specialized functions?

35. Evolution of gene regulation – Why? • Prokaryotes • single-celled • evolved to grow & divide rapidly • must respond quickly to changes in external environment • exploit transient resources – lac and trp operons • Gene regulation • turn genes on & off rapidly • flexibility & reversibility • adjust levels of enzymes for synthesis & digestion

36. Evolution of gene regulation • Eukaryotes • multicellular • evolved to maintain constant internal conditions while facing changing external conditions • homeostasis • regulate body as a whole • growth & development • long term processes • specialization • turn on & off large number of genes - #18 • 20% of genes are turned on in a typical cell - # 19 • must coordinate the body as a whole rather than serve the needs of individual cells

37. Points of control • The control of gene expression can occur at any step in the pathway from gene to functional protein 1. packing/unpacking DNA 2. Transcription (common to all organisms # 20) 3. mRNA processing 4. mRNA transport 5. translation 6. protein processing 7. protein degradation

38. 1. DNA packing How do you fit all that DNA into nucleus? • DNA coiling & folding • double helix • nucleosomes • chromatin fiber • looped domains • chromosome from DNA double helix to condensed chromosome

39. 8 histone molecules Nucleosomes • “Beads on a string” • 1st level of DNA packing • histone proteins • 8 protein molecules • positively charged amino acids • bind tightly to negatively charged DNA DNA packing movie

40. DNA packing as gene control (Epigenetic inheritance) - # 21 • Degree of packing of DNA regulates transcription • tightly wrapped around histones • no transcription • genes turned off • heterochromatin darker DNA (H) = tightly packed • euchromatin lighter DNA (E) = loosely packed H E

41. #22 - Histone acetylation • Acetylation of histones unwinds DNA • loosely wrapped around histones • enables transcription • genes turned on • attachment of acetyl groups (–COCH3) to histones • conformational change in histone proteins • transcription factors have easier access to genes

42. #23 - DNA methylation • Methylation of DNA blocks transcription factors • no transcription  genes turned off • attachment of methyl groups (–CH3) to cytosine • C = cytosine • nearly permanent inactivation of genes • #24 - ex. inactivated mammalian X chromosome = Barr body • Can be passed down to new cells

43. #25 - Genomic Imprinting • When there is phenotypic variation due to which parent gave a certain gene (not sex-linked, found on autosomes) • 1 allele is silenced by DNA methylation • Only 1 copy of certain genes is needed for normal development • If 0 or 2 genes is active, then major developmental problems can occur

44. 2. Transcription initiation • Control regions on DNA • promoter • nearby control sequence on DNA • binding of RNA polymerase & transcription factors • “base” rate of transcription • enhancer • distant control sequences on DNA • binding of activator proteins • “enhanced” rate (high level) of transcription

45. Classes of Transcription Factors • General – produce a low rate of transcription, needed for all genes • Specific – interact with specific control elements (upstream of the gene) – greatly increases transcription rate

46. Model for Enhancer action • Enhancer DNA sequences • distant control sequences • Activator proteins • bind to enhancer sequence & stimulates transcription • Silencer proteins • bind to enhancer sequence & block gene transcription Turning on Gene movie

47. Transcription complex Activator Proteins • regulatory proteins bind to DNA at distant enhancer sites • increase the rate of transcription Enhancer Sites regulatory sites on DNA distant from gene Enhancer Activator Activator Activator Coactivator E F B RNA polymerase II H TFIID A Coding region T A T A Core promoter and initiation complex Initiation Complex at Promoter Site binding site of RNA polymerase

48. Signal transduction pathways Signal molecule can: Bind to cell surface Cause activator or repressor to be turned on/off Diffuse through the cell Bind to an activator or repressor to be turned on/off

49. #28 - Prokaryotic vs Eukaryotic Transcriptional control • Bacteria have operons regulated by 1 promoter • Eukaryotes have related genes on different chromosomes with different promoters • Eukaryotes have control elements for each gene • Control elements – specific nucleotide sequences, unique combination for each gene

50. #29 - Coordinated control of genes in eukaryotes • Related genes are clustered together • Chromatin structure of that area is manipulated • Turns all genes in that area on or off • Related genes have the same combination of control elements • When those control elements are present, all related genes are turned on