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Bio 101A

Bio 101A. GE III results day. Which one of the following occurs when RNA polymerase attaches to the promoter DNA?   A) elongation of the growing RNA molecule   B) termination of the RNA molecule   C) addition of nucleotides to the DNA template   D) initiation of a new RNA molecule  

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Bio 101A

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  1. Bio 101A GE III results day

  2. Which one of the following occurs when RNA polymerase attaches to the promoter DNA?   A) elongation of the growing RNA molecule   B) termination of the RNA molecule   C) addition of nucleotides to the DNA template   D) initiation of a new RNA molecule   E) initiation of a new polypeptide chain

  3. Which of the following is the enzyme that HIV uses to synthesize DNA on an RNA template?   A) ligase   B) RNA polymerase   C) terminator enzyme   D) reverse transcriptase   E) None of the choices are correct.

  4. Prokaryotic Gene Regulation Bio 101A • Operon structure and function • Positive/Negative gene regulation • Promoters, operators, repressors, inducers

  5. Q: What is “regulation”? "When I was warning about the danger ahead on Wall Street months ago because of the lack of oversight, Senator McCain was telling the Wall Street Journal -- and I quote -- 'I'm always for less regulation.' " – Sen. Barack Obama “Senator Obama was silent on the regulation of Fannie Mae and Freddie Mac, and his Democratic allies in Congress opposed every effort to rein them in…last year he said that subprime loans had been, quote, “a good idea.””- Sen. John McCain

  6. Enzymes are coded for by genes • DNA is the code to make proteins • Enzymes are made of protein • In order for a cell to make an enzyme, it must access the DNA for that enzyme • Enzymes are very specific to their task

  7. V. fischeri interacts symbiotically with a squid • Helps the squid camouflage itself during nocturnal hunting • 95% of colonies are expelled daily • The rest are fed in pouches in the squid’s tissue • Bacterium has an interest in regulating expression of luciferase gene

  8. V. fischeri interacts symbiotically with a squid The winnowing: establishing the squid–vibrio symbiosis Spencer V. Nyholm & Margaret McFall-Ngai Nature Reviews Microbiology 2, 632-642 (August 2004)

  9. Enzyme Regulation maintains Efficiency • Enzymes: General Background b • - galactosidase • Gene Regulation • Biochemical Regulation • Experiment 9

  10. b - galactosidase 10

  11. b - galactosidase H O 2 galactose lactose b - galactosidase glucose (aka lactase in humans) 11

  12. b - galactosidase Regulation b Why Regulate - galactosidase ? • b Levels at which - galactosidase can be • regulated: Genetic • Biochemical • 12

  13. The lac operon regulates when β-galactosidase is made

  14. When lactose is present, transcription is activated

  15. Regulation of enzyme production Regulation of enzyme activity Precursor Feedback inhibition Enzyme 1 Gene 1 LE 18-20 Gene 2 Enzyme 2 Regulation of gene expression Gene 3 Enzyme 3 Enzyme 4 Gene 4 Gene 5 Enzyme 5 Tryptophan

  16. Operons: The Basic Concept • An operon is a collection of prokaryotic genes transcribed together on a single mRNA transcript to serve a single purpose • Composed of • An operator, an “on-off” switch • A promoter • Genes for metabolic enzymes • Can be switched off by a repressor protein • A corepressor is a small molecule that binds to a repressor to switch an operon off

  17. trp operon Promoter Promoter Prokaryotic Operon structure ensures efficient regulation of transcription Genes of operon DNA trpB trpA trpE trpC trpD trpR Operator Stop codon RNA polymerase Regulatory gene Start codon 3¢ mRNA 5¢ mRNA 5¢ D B E C A Inactive repressor Protein Polypeptides that make up enzymes for tryptophan synthesis Tryptophan absent, repressor inactive, operon on The tryptophan biosynthesis operon is repressible by the presence of its product, tryptophan

  18. DNA LE 18-21b_1 mRNA Active repressor Protein Tryptophan (corepressor) Tryptophan present, repressor active, operon off

  19. DNA No RNA made LE 18-21b_2 mRNA Active repressor Protein Tryptophan (corepressor) Tryptophan present, repressor active, operon off

  20. Repressor Protein Repressor mRNA Promoter Operator Structural Genes RNA Polymerase Regulator Gene Basic Operon Regulation NO TRANSCRIPTION

  21. Tryptophan Operon Tryptophan Present Regulator Gene Promoter Operator Attenuator Structural Genes RNA Polymerase NO TRANSCRIPTION trpRmRNA Q: Why might the cell want to produce an aporepressor that is only activated by the operon’s end product? + tryptophan (corepressor) TrpR protein (homodimer) TrpR aporepressor + corepressor (can bind to operator)

  22. Tryptophan Operon Tryptophan Absent Regulator Gene Promoter Operator Attenuator Structural Genes RNA Polymerase TRANSCRIPTION trpRmRNA TrpR protein (homodimer) TrpR aporepressor (cannot bind to operator)

  23. Tryptophan Repressor Protein TrpR protein subunits Tryptophan (co-repressor) DNA

  24. Repressible and Inducible Operons: Two Types of Negative Gene Regulation • A repressible operon is one that is usually on; binding of a repressor shuts off transcription • The trp operon is a repressible operon • An inducible operon is one that is usually off; a molecule called an inducer inactivates the repressor and turns on transcription • The classic example of an inducible operon is the lac operon

  25. Promoter Regulatory gene Operator lacl lacZ DNA LE 18-22a No RNA made 3¢ mRNA RNA polymerase 5¢ Active repressor Protein Lactose absent, repressor active, operon off

  26. lac operon DNA lacl lacY lacA lacZ LE 18-22b RNA polymerase 3¢ mRNA mRNA 5¢ 5¢ Transacetylase Permease -Galactosidase Protein Inactive repressor Allolactose (inducer) Lactose present, repressor inactive, operon on

  27. Inducible enzymes usually function in catabolic pathways • Repressible enzymes usually function in anabolic pathways • Regulation of the trp and lac operons involves negative control of genes because operons are switched off by the active form of the repressor

  28. Positive Gene Regulation • Some operons are also subject to positive control through a stimulatory activator protein, such as catabolite activator protein (CAP) • When glucose (a preferred food source of E. coli ) is scarce, the lac operon is activated by the binding of CAP • When glucose levels increase, CAP detaches from the lac operon, turning it off

  29. Promoter DNA lacl lacZ LE 18-23a RNA polymerase can bind and transcribe Operator CAP-binding site Active CAP cAMP Inactive lac repressor Inactive CAP Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized

  30. Promoter DNA lacl lacZ LE 18-23b CAP-binding site Operator RNA polymerase can’t bind Inactive CAP Inactive lac repressor Lactose present, glucose present (cAMP level low): little lac mRNA synthesized

  31. In a prokaryote, a group of genes with related functions, along with their associated control sequences, defines   A) an allele.   B) an operon.   C) a locus.   D) a transposon.   E) a chromosome.  

  32. mRNA β-gal Operons can be cut and pasted together to make operon fusions Tryptophan Operon Lactose Operon Repressor lacI T Pro. Oper. Z gene Y gene A gene Pro. Oper. TrpE, D, C, B, A Att. Operator Promoter Z gene Y gene A gene mutant trpR-containing plasmid

  33. mRNA β-gal If the repressor is knocked out, what will happen in the presence of Tryptophan? Tryptophan Operon Lactose Operon Repressor lacI T Pro. Oper. Z gene Y gene A gene Pro. Oper. TrpE, D, C, B, A Att. Operator Promoter Z gene Y gene A gene

  34. mRNA β-gal What if we add a plasmid which contains the TrpR gene? With tryptophan? Without? Tryptophan Operon Lactose Operon Repressor lacI T Pro. Oper. Z gene Y gene A gene Pro. Oper. TrpE, D, C, B, A Att. Operator Promoter Z gene Y gene A gene mutant trpR-containing plasmid

  35. Another engineered plasmid with fusion Operon: pGLO araC ori pGLO GFP bla • Manufactured by a private corporation • AraC- arabinose gene • GFP- Green Fluorescent protein • bla- Beta-lactamase • ori- you know this… VandePol

  36. Is this:Anabolic or Catabolic?Positive or negative?Inducible or repressible?

  37. Expression of Green Fluorescent Protein • How do you think this fusion was made? • What are the structural sequences? The regulatory sequences? • What happens when we add arabinose sugar to these bacteria? • What do you think is meant by “reporter gene”?

  38. lac Operon and ara operon lac Operon ara Operon araC LacI Z Y A B A D Effector (Lactose) Effector (Arabinose) araC B A D LacI Z Y A RNA Polymerase B A D Z Y A araC RNA Polymerase

  39. Which colonies will glow? LB/Amp LB/Amp/Ara LB Grow? Glow? • Follow protocol • On which plates will colonies grow? • Which colonies will glow?

  40. Appendix: pGLO slides that may be helpful • Stuff about GFP, arabinose, beta-lactamase, etc.

  41. DNA polymerase binds to the ori Parental (template) strand 0.25 µm Origin of replication Daughter (new) strand LE 16-12 Replication fork Bubble Two daughter DNA molecules In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM). In eukaryotes, DNA replication begins at may sites along the giant DNA molecule of each chromosome.

  42. ara GFP Operon ara Operon araC GFP Gene araC B A D Effector(Arabinose) Effector (Arabinose) araC B A D araC GFP Gene RNA Polymerase RNA Polymerase B A D araC araC GFP Gene Gene Regulation On pGLO, the regulatory regions of the Arabinose operon have been glued to the structural sequences for GFP What will happen on the Ara (+) plates? What will happen on the Ara (-) plates?

  43. 40 Å 30 Å Green Fluorescent Protein • discovered in 1960s by Dr. Frank Johnson and colleagues • closely related to jellyfish aequorin • absorption max = 470nm • emission max = 508nm • 238 amino acids, 27kDa • “beta can” conformation: 11 antiparallel beta sheets, 4 alpha helices, and a centered chromophore • amino acid substitutions result in several variants, including YFP, BFP, and CFP

  44. Using GFP as a biological tracer GFP can be fused to cellular proteins http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html With permission from Marc Zimmer

  45. Control Region Gene X (no stop codon) GFP gene (in-frame with Gene X) express gene-of-interest GFP Fusion & Protein Localization Gene Fusion 1 Translated Fusion Protein Fluorescence Visualization 2 3 GFP Protein X

  46. Fusion protein in C. elegans highlights nervous system Vesicle Transport in the Caenorhabditis elegans Nervous System

  47. GFP Synaptobrevin SNB-1::GFP Fusion Fusion of snb-1 to GFP gene 1 Control Region For snb-1 GFP gene (in-frame with snb-1) express gene-of-interest Neurotransmitter-Packaging Vesicles Present GFP-Tagged SNB-1 on Surface View SNB-1 (Vesicle) Distribution 2 3

  48. 2008 Nobel Prize- GFP • GFP mice

  49. The pGLO plasmid • ori- origin of replication • GFP- green fluorescent protein • bla- Beta-lactamase • araC- Arabinose • What are all the other marks? Why are they there?

  50. Beta- lactam antibiotics have a similar structure • Includes penicillin, ampicillin, and others • The beta-lactam ring is a square structure common to all

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