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  • If one enzyme responsible for each step, should be able to find spores deficient in each enzyme. Normal Neurospora can produce own arginine, therefore grows when arginine not present in the environment (i.e. In minimal medium) Used X rays to mutate Neurospora Found colonies that could grow when provided with arginine, but did not grow when arginine was not present these must have a deficiency of one of the enzymes in the metabolic pathway for producing arginine Each mutant required a specific nutrient, the nutrient that was required indicated which enzyme was malfunctioning
  • If one enzyme responsible for each step, should be able to find spores deficient in each enzyme. Normal Neurospora can produce own arginine, therefore grows when arginine not present in the environment (i.e. In minimal medium) Used X rays to mutate Neurospora Found colonies that could grow when provided with arginine, but did not grow when arginine was not present these must have a deficiency of one of the enzymes in the metabolic pathway for producing arginine Each mutant required a specific nutrient, the nutrient that was required indicated which enzyme was malfunctioning
  • If one enzyme responsible for each step, should be able to find spores deficient in each enzyme. Normal Neurospora can produce own arginine, therefore grows when arginine not present in the environment (i.e. In minimal medium) Used X rays to mutate Neurospora Found colonies that could grow when provided with arginine, but did not grow when arginine was not present these must have a deficiency of one of the enzymes in the metabolic pathway for producing arginine Each mutant required a specific nutrient, the nutrient that was required indicated which enzyme was malfunctioning
  • If one enzyme responsible for each step, should be able to find spores deficient in each enzyme. Normal Neurospora can produce own arginine, therefore grows when arginine not present in the environment (i.e. In minimal medium) Used X rays to mutate Neurospora Found colonies that could grow when provided with arginine, but did not grow when arginine was not present these must have a deficiency of one of the enzymes in the metabolic pathway for producing arginine Each mutant required a specific nutrient, the nutrient that was required indicated which enzyme was malfunctioning
  • If one enzyme responsible for each step, should be able to find spores deficient in each enzyme. Normal Neurospora can produce own arginine, therefore grows when arginine not present in the environment (i.e. In minimal medium) Used X rays to mutate Neurospora Found colonies that could grow when provided with arginine, but did not grow when arginine was not present these must have a deficiency of one of the enzymes in the metabolic pathway for producing arginine Each mutant required a specific nutrient, the nutrient that was required indicated which enzyme was malfunctioning
  • If one enzyme responsible for each step, should be able to find spores deficient in each enzyme. Normal Neurospora can produce own arginine, therefore grows when arginine not present in the environment (i.e. In minimal medium) Used X rays to mutate Neurospora Found colonies that could grow when provided with arginine, but did not grow when arginine was not present these must have a deficiency of one of the enzymes in the metabolic pathway for producing arginine Each mutant required a specific nutrient, the nutrient that was required indicated which enzyme was malfunctioning
  • Point out Genes A, B, C lead to the enzymes
  • Transcription

    1. 1. TranscriptionChapter 17: page 303-313Historical ExperimentsMechanismProcessing
    2. 2. Contents Historical Experiments  Garrod  Beadle & Tatum Transcription Mechanism  Initiation, elongation, termination RNA processing  post-transcriptional modifications:  5’ cap  Poly A tail  splicing
    3. 3. What did they think of inheritance in the 1900s?  Only physical traits were inherited  Example: hair colour, height etc.http://www.toonpool.com/user/8371/files/long_face_1055695.jpg http://www.lab-initio.com/250dpi/nz251.jpg
    4. 4. Archibald Garrod (1909) British physician First to suggest a link between inheritance (genes) and its effect on a chemical reaction in the cell (metabolic pathway) leading to observable symptoms (physical traits) “inborn errors of metabolism” Genes  enzymes  phenotype Genes are not just responsible for passing on traditional inherited traits Used disease alkaptonuria in his reasoning http://www.nature.com/scitable/content/6994/pierce_3_garrod_FULL.jpg
    5. 5. Alkaptonuria a condition where urine is black due to build up of homogentisic acid (alkapton) alkapton darkens when exposed to air http://www.nature.com/scitable/content/6994/pierce_3_garrod_FULL.jpg
    6. 6. Garrod’s Postulates Hypothesis specific to alkaptonuria:  Individuals with alkaptonuria lack an enzyme that breakdown alkapton  no enzyme  alkaptonuria Hypothesis generalized from alkaptonuria:  Symptoms of an inherited disease is due to the inability to make an enzyme  inheritance  no enzyme  symptoms Assumptions that stems from generalization:  Genes dictate phenotypes through enzymes that catalyze specific reactions in the cell  genes  enzymes  phenotype
    7. 7. A Metabolic Pathway hypothesis was based on the idea that chemical reactions take place in a series of steps (metabolic pathway) But his hypothesis was untested
    8. 8. Alkaptonuria now(This slide is for interest only. No need to study it.) Malfunction in the tyrosine catabolic pathway Enzyme homogentisate dioxygenase doesn’t work Buildup of homogentisate X http://upload.wikimedia.org/wikipedia/commons/thumb/c/c9/Tyrosinedegradation2.png/750px-Tyrosinedegradation2.png
    9. 9. George Beadle andEdward Tatum (1930s)  Won Nobel prize in 1958  Performed research based Garrod’s ideas  Proposed that genes were responsible for producing enzymes  “One gene one enzyme hypothesis” http://nobelprize.org/nobel_prizes/medicine/laureates/1958/tatum.jpg http://nobelprize.org/nobel_prizes/medicine/laureates/1958/beadle.jpg
    10. 10. Beadle & Tatum Experiment Experimented using the bread mold Neurospora crassa Simple organism  Short life cycle  Require access to only a few biological substances to live and grow (salts, sugar and vitamin B)
    11. 11. Beadle & Tatum Experiment Hypothesized that mold:  must be able to synthesize their own amino acids  have enzymes that convert the simple substances into amino acids needed for growth To test hypothesis:  mutate the genes in mold  look for mutations that affected the mold’s ability to make amino acids
    12. 12. Step 1: Making mutants Treated mold with x-rays to mutate the DNA  1927 Herman Muller showed that x-rays cause mutation in genes Looked for mutations that prevented mold from synthesizing arginine  Arginine (arg) is an amino acid
    13. 13. Step 2: Isolating arg mutantsMold strain Wildtype* Arginine mutantComplete MinimalMinimal +arginine *Wildtype: unmutated, normal
    14. 14. Step 2: Isolating arg mutants
    15. 15. Step 2: Isolating arg mutants Media Description of media Contents of media Everything needed for salt, sugar, vitamin B Complete growth without having and all amino acids to synthesize it Only has what is salt, sugar, vitamin B Minimal essential* for growth Only has what is salt, sugar, vitamin B Minimal + essential* for growth and arginineone nutrient plus one nutrient *Essential: required for survival but cannot be made by the organism, thus must be provided externally
    16. 16. Step 3: Isolating mutation inarginine metabolic pathway Studied the synthesis of arginine in bread mold
    17. 17. Beadle & Tatum Rationale Mutants that did not grow in minimal media but grew with the nutrient meant that the pathway responsible for making the nutrient was “broken” Thus the malfunctioning enzyme was due to mutations in genes Thus genes are responsible for enzyme production
    18. 18. Situation A: arg1 mutant Enzyme Enzyme Enzyme A B CGiven: Given: Given: Given:MM + Precursor MM + Ornithine MM + Citrulline MM + Argininedoes not grow grows grows grows
    19. 19. Situation B: arg2 mutant Enzyme Enzyme Enzyme A B CGiven: Given: Given: Given:MM + Precursor MM + Ornithine MM + Citrulline MM + Argininedoes not grow does not grow grows grows
    20. 20. Situation C: arg3 mutant Enzyme Enzyme Enzyme A B CGiven: Given: Given: Given:MM + Precursor MM + Ornithine MM + Citrulline MM + Argininedoes not grow does not grow does not grow grows
    21. 21. Beadle & Tatum Experiment http://biology.kenyon.edu/courses/biol114/Chap04/Chapter_04.html
    22. 22. Beadle & Tatum Experiment
    23. 23. Beadle & Tatum Conclusion Showed how genes are related to proteins  Each mutant was defective in a single gene  Mutations in the genes affected production of enzymes in metabolic pathways  Implied that metabolic diseases can be inherited One gene, one enzyme hypothesis: The function of a gene is to dictate the production of a specific enzyme
    24. 24. Modification to hypothesis One gene, one protein  Not all enzymes are proteins  Some enzymes are RNA (ribozymes) One gene, one polypeptide  One protein may have more than one subunit Other exceptions:  Some genes do not produce polypeptide (regulatory: e.g. 5’ UTR)  One gene may produce multiple polypeptide (due to splicing)
    25. 25. Beadle & Tatum Video Tutorial http://www.dnalc.org/view/16360-Animation-16-One-gene-ma http://wps.prenhall.com/wps/media/objects/1552/1589869 /web_tut/21_04/21_04_01a.swf Discovery Channel 100 Greatest Discoveries – History of Genetics (Beadle & Tatum @ 8:47-11:10) http://www.youtube.com/watch?v=0qgMd0obEkc
    26. 26. Central Dogma Location Process DNA Nucleus Transcription mRNA Cytoplasm or RER Translation protein Animation: http://www.stolaf.edu/people/giannini/flashanimat/molgenetics/transcription.swf Animation: http://www.stolaf.edu/people/giannini/flashanimat/molgenetics/translation.swf
    27. 27. Central Dogma Animations Clip from PBS “DNA: The Secret of Life” (4:06) http://www.youtube.com/wat ch?v=41_Ne5mS2ls Tutorial http://www.wiley.com/legacy/ college/boyer/0470003790/ani mations/central_dogma/centr al_dogma.htm
    28. 28. Gene  The stretch of DNA that is transcribed into RNA  Also called a transcription unit upstream downstream promoter transcription unit termination sequence start transcription +1negative numbers positive numbers
    29. 29. Gene As you learn about the mechanism of transcription and translation determine whether the following parts are part of the gene:  Introns and exons  5’ cap and poly A tails  DNA that codes for the start codon  DNA that codes for the stop codon  5’ leader sequence  3’ trailer sequence
    30. 30. Stages of Transcription Initiation Elongation Termination Fig. 17.6a
    31. 31. Promoter Where RNA polymerase (RNAP) first binds Prokaryote has one RNAP Eukaryote has three:  RNAP I makes rRNA  RNAP II involved in transcription (makes mRNA)  RNAP III makes tRNA Fig. 17.7
    32. 32. Promoter Located upstream of the gene Composed of an AT rich region  Why is this region rich in A & T? Hint: DNA is double stranded AT Rich Region Organism Location Consensus in gene sequence Pribnow box Prokaryote -10 TATAAT TATA box Eukaryote -25 TATAAA Fig. 17.7
    33. 33. Promoter Contains the transcription start point location where RNAP starts transcribing Fig. 17.7
    34. 34. Transcription Initiation Prokaryote: RNAP recognize and bind to promoter Eukaryote: transcription factors bind first, then RNAP II Transcription initiation complex: TF + RNAP on promoter Fig. 17.7
    35. 35. Transcription Factors protein that are involved in starting transcription some help control how often genes are transcribed
    36. 36. Defining the strands Coding strand = sense strand Noncoding strand = antisense strand = template Transcript = newly synthesized RNA
    37. 37. Practice In this diagram identify the following:  Transcript  Template strand  Coding strand  Sense strand  Antisense strand Label the 5’ and 3’ end on each strand
    38. 38. Transcription Elongation RNAP unwinds DNA exposing 10-20 bases Uses template strand to add complementary RNA nucleotides Direction of transcription: downstream from 5’3’ (opposite is upstream) Fig. 17.6b
    39. 39. Transcription Elongation RNA transcript separates from template as RNAP continues elongation DNA helix reforms behind RNAP Many RNAP can transcribe simultaneously Fig. 17.6b
    40. 40. Transcription Termination Terminator sequence: signals end of transcription  Signal is actually the RNA sequence (transcribed terminator)  Eukaryote: AAUAAA Fig. 17.8
    41. 41. Transcription Termination Prokaryote: transcription ends immediately at signal Eukaryote:  RNAP continues for hundreds of nucleotides past termination signal  at 10-35 nucleotide past signal pre-mRNA is released Fig. 17.8
    42. 42. RNA Processing Pre-mRNA = raw mRNA = primary transcript: mRNA before RNA is processed RNA processing only occurs in eukaryotes Pre-mRNA = raw mRNA = primary transcript Fig. 17.8
    43. 43. RNA Processing Post-transcriptional modification:  Capping: 5’ cap  Polyadenylation: Poly A tail Splicing: intron excision
    44. 44. Post-TranscriptionalModification: Capping 5’ cap  Modified guanine (7-methylguanosine triphosphate) added to 5’ end  Added by capping enzyme complex Function:  Protect mRNA from degradation  Signals ribosome attachment
    45. 45. 5’ cap
    46. 46. Post-TranscriptionalModification: Polyadenylation PolyA tail  50-250 adenine added to 3’ end  Added by poly-A polymerase Function  Protect mRNA from degradation  Facilitate export of mRNA from nucleus
    47. 47. RNA Processing: Splicing  Splicing: removal of introns  Introns: intervening (noncoding) sequences, interspersed between exons  Exons: coding sequences, expressedFig. 17.9
    48. 48. RNA Processing: Splicing  Exception to ‘exon is expressed’ rule  Leader = 5’ untranslated region = 5’ UTR  Trailer = 3’ untranslated region = 3’ UTR  Exon sequence that is untranslatedFig. 17.9
    49. 49. Splicing VideoGeneral intro to splicing: http://www.dnalc.org/resources/3 d/24-mrna-splicing.htmlSplicing mechanism: http://highered.mcgraw- hill.com/olc/dl/120077/bio30.swf http://www.dnalc.org/resources/3 d/rna-splicing.html
    50. 50. Splice Site short nucleotide sequences at ends of introns where snRNPs bind and thus where excision occurs Fig. 17.10
    51. 51. snRNP “snurps” Small nuclear ribonucleoprotein (composed of snRNA and protein) Ribozyme Fig. 17.10
    52. 52. Mechanism of Splicing snRNP bind to splice site Form a splicesome:  snRNP + other proteins Excises intron Rejoins exons Fig. 17.10
    53. 53. Co-TranscriptionalSplicing Proudfoot, N. (2000). Connecting transcription to messenger RNA processing. Trends in Biochemical Sciences, 25(6), 290-293. Fig. 2. Electron micrograph directly visualizing co- transcriptional splicing. The gene shown is from Drosophila and is ∼6 kb long. (Photograph courtesy of Ann Beyer and Yvonne Osheim, University of Virginia, VA, USA.) DNA enters at the upper left of the micrograph and exits at the lower left. Transcription initiates near the position marked with an asterisk; nascent RNA transcripts appear as fibrils of increasing length that emanate from the DNA template. The transcripts are undergoing co-transcriptional splicing, as indicated by the progressive formation and loss of intron loops of various sizes near the 5’ ends of the transcripts (arrows). The large arrow indicates a transcript near the 3’ end of the gene that is no longer attached to the DNA template and might have been caught in the act of termination and release. 200 nm http://ars.els-cdn.com/content/image/1-s2.0-S0968000400015917-gr2.jpg
    54. 54. Intron Function Regulatory role Alternative splicing: single gene encode more than one kind of polypeptide depending on which segments are treated as exons http://www.realscience.org.uk/pics/alternative_splicing2.gif
    55. 55. Intron Function Exon shuffling: Introns increase probability of beneficial crossing over, recombination between two alleles  change protein domains without affecting coding sequences Fig. 17.11
    56. 56. Prokaryotic versus Eukaryotic protein synthesis Video: http://highered.mcgraw- hill.com/olc/dl/120077/bio25.swf Fig. 17.2ab
    57. 57. HW Questions Complete RNA Processing Worksheet Which enzymes in DNA replication mimic the function of RNAP in initiation and elongation? Why would promoters have a higher abundance of A-T than G-Cs? Why is post-transcriptional modifications not necessary in prokaryotes? Summarize in a chart, all the differences between prokaryotes and eukaryotes in transcription

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