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Ch. 17:From Gene to Protein

Discover how genes work, what they code for, and how genetic information flows from DNA to proteins. Explore transcription, translation, and the importance of proteins in cellular function and disease. Learn about the one gene-one enzyme hypothesis and the role of genes in metabolic diseases.

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Ch. 17:From Gene to Protein

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  1. Ch. 17:From Gene to Protein How Genes Work

  2. What do genes code for? • How does DNA code for cells & bodies? • how are cells and bodies made from the instructions in DNA DNA proteins cells bodies

  3. The “Central Dogma” • Flow of genetic information in a cell • How do we move information from DNA to proteins? transcription translation RNA DNA protein trait DNA gets all the glory, but proteins do all the work! replication

  4. A B C D E disease disease disease disease Metabolism taught us about genes • Inheritance of metabolic diseases • suggested that genes coded for enzymes • each disease (phenotype) is caused by non-functional gene product • lack of an enzyme • Tay sachs • PKU (phenylketonuria) • albinism Am I just the sum of my proteins? metabolic pathway     enzyme 1 enzyme 2 enzyme 3 enzyme 4

  5. 1941 | 1958 Beadle & Tatum one gene : one enzyme hypothesis George Beadle Edward Tatum "for their discovery that genes act by regulating definite chemical events"

  6. RESULTS EXPERIMENT Classes of Neurospora crassa Growth:Wild-typecells growingand dividing No growth:Mutant cellscannot growand divide Figure 17.2 Wild type Class III mutants Class II mutants Class I mutants Minimalmedium(MM) (control) Minimal medium MM ornithine X Beadle & Tatum Does one gene Control one trait? Condition MM citrulline X MM arginine(control) X Must be suppliedornithine,citrulline, orarginine Can grow withor without anysupplements Must be supplied citrulline orarginine Summaryof results Must be supplied arginineto grow CONCLUSION Class I mutants(mutation ingene A) Gene (codes forenzyme) Class II mutants(mutation ingene B) Class III mutants(mutation ingene C) Wild type Precursor Precursor Precursor Precursor Gene A Enzyme A Enzyme A Enzyme A Enzyme A Ornithine Ornithine Ornithine Ornithine Gene B Enzyme B Enzyme B Enzyme B Enzyme B Citrulline Citrulline Citrulline Citrulline Gene C Enzyme C Enzyme C Enzyme C Enzyme C Arginine Arginine Arginine Arginine

  7. aa aa aa aa aa ribosome aa aa aa aa aa aa From gene to protein nucleus cytoplasm transcription translation DNA mRNA protein trait

  8. Transcription fromDNA nucleic acid languagetoRNA nucleic acid language

  9. RNA • ribose sugar • N-bases • uracil instead of thymine • U : A • C : G • single stranded • lots of RNAs • mRNA, tRNA, rRNA, siRNA… transcription DNA RNA

  10. Transcription • Making mRNA • transcribed DNA strand = template strand • untranscribed DNA strand = coding strand • same sequence as RNA • synthesis of complementary RNA strand • transcription bubble • enzyme • RNA polymerase coding strand 3 A G C A T C G T 5 A G A A A C G T T T T C A T C G A C T DNA 3 C T G A A 5 T G G C A U C G U T C unwinding 3 G T A G C A rewinding mRNA template strand RNA polymerase 5 build RNA 53

  11. RNA polymerases • 3 RNA polymerase enzymes • RNA polymerase 1 • only transcribes rRNA genes • makes ribosomes • RNA polymerase 2 • transcribes genes into mRNA • RNA polymerase 3 • only transcribes tRNA genes • each has a specific promoter sequence it recognizes

  12. Which gene is read? • Promoter region • binding site before beginning of gene • TATA box binding site • binding site for RNA polymerase & transcription factors • Enhancer region • binding site far upstream of gene • turns transcription on HIGH

  13. Transcription Factors • Initiation complex • transcription factors bind to promoter region • suite of proteins which bind to DNA • hormones? • turn on or off transcription • trigger the binding of RNA polymerase to DNA

  14. RNA polymerase Matching bases of DNA & RNA A • Match RNA bases to DNA bases on one of the DNA strands C U G A G G U C U U G C A C A U A G A C U A 5' 3' G C C A T G G T A C A G C T A G T C A T C G T A C C G T

  15. intron = noncoding (inbetween) sequence exon = coding (expressed) sequence Eukaryotic genes have junk! • Eukaryotic genes are not continuous • exons = the real gene • expressed / coding DNA • introns = the junk • inbetween sequence intronscome out! eukaryotic DNA

  16. intron = noncoding (inbetween) sequence exon = coding (expressed) sequence mRNA splicing • Post-transcriptional processing • eukaryotic mRNA needs work after transcription • primary transcript = pre-mRNA • mRNA splicing • edit out introns • make mature mRNA transcript ~10,000 bases eukaryotic DNA pre-mRNA primary mRNA transcript ~1,000 bases mature mRNA transcript spliced mRNA

  17. 1977 | 1993 Discovery of exons/introns Richard Roberts Philip Sharp adenovirus CSHL MIT common cold beta-thalassemia

  18. Oops! This trait Won’t be expressed! Sequence must be accurate • No room for mistakes! • a single base added or lost throws off the reading frame AUG-CGU-UCU-GAU-AAA-GGU-CAC-… AUG-CGG-UCC-GAC-AAG-GGC-CAU-… AUG-CGC-UCA-GAU-AAG-GGG-CAC-… Met-Arg-Ser-Asp-Lys-Gly-His-… AUG-CGU-GUC-(U-GA)(U-AA)(A-GG)(U-CA)(C-… Met – Arg- Val- STOP

  19. snRNPs snRNA intron exon exon 5' 3' spliceosome 5' 3' lariat 5' 3' exon exon mature mRNA excised intron 5' 3' Whoa! I think we just brokea biological “rule”! RNA splicing enzymes • snRNPs • small nuclear RNA • proteins • Spliceosome • several snRNPs • recognize splice site sequence • cut & paste gene No, not smurfs! “snurps”

  20. Alternative splicing • Alternative mRNAs produced from same gene • when is an intron not an intron… • different segments treated as exons • different traits from one mRNA One gene can code for more than one trait? So, it’s not just junk DNA!

  21. enhancer translation start translation stop exons 1000+b 20-30b RNA polymerase DNA UTR UTR introns promoter transcription start transcription stop pre-mRNA 5' 3' 5' 3' mature mRNA The Transcriptional unit (gene or genes?) transcriptional unit (gene) 3' 5' TAC ACT TATA DNA GTP AAAAAAAA

  22. 3' poly-A tail 3' A A A A A mRNA 50-250 A’s 5' cap P P P 5' G More post-transcriptional processing • Need to protect mRNA on its trip from nucleus to cytoplasm • enzymes in cytoplasm attack mRNA • protect the ends of the molecule • add 5 GTP cap • add poly-A tail • longer tail, mRNA lasts longer: produces more protein So, these enzymes are like a dog…give it a bone to chew on so it doesn’t chew your shoes! http://vcell.ndsu.edu/animations/transcription/movie-flash.htm

  23. aa aa aa aa aa ribosome aa aa aa aa aa aa From gene to protein nucleus cytoplasm transcription translation DNA mRNA protein trait

  24. mRNA Transcription in real time. http://www.youtube.com/watch?v=5MfSYnItYvg The Central Dogma http://www.youtube.com/watch?v=J3HVVi2k2No

  25. Translation fromnucleic acid languagetoamino acid language

  26. TACGCACATTTACGTACGCGG DNA AUGCGUGUAAAUGCAUGCGCC mRNA MetArgValAsnAlaCysAla protein ? How does mRNA code for proteins? How can you code for 20 amino acids with only 4 nucleotide bases (A,U,G,C)? 4 ATCG 4 AUCG 20

  27. mRNA codes for proteins in triplets TAC-GCA-CAT-TTA-CGT-ACG-CGG DNA DNA codon mRNA AUG-CGU-GUA-AAU-GCA-UGC-GCC protein Met-Arg–Val–Asn-Ala-Cys-Ala

  28. 1960 | 1968 Cracking the code Nirenberg & Khorana • Crick • determined 3-letter (triplet) codon system WHYDIDTHEREDBATEATTHEFATRAT WHYDIDTHEREDBATEATTHEFATRAT • Nirenberg (47) & Khorana (17) • determined mRNA–amino acid match • added fabricated mRNA to test tube of ribosomes, tRNA & amino acids • created artificial UUUUU… mRNA • found that UUU coded for phenylalanine

  29. 1960 | 1968 Marshall Nirenberg Har Khorana

  30. The code • Code for ALL life! • strongest support for a common origin for all life • Code is redundant • several codons for each amino acid • 3rd base “wobble” Why is thewobble good? • Start codon • AUG • methionine • Stop codons • UGA, UAA, UAG

  31. aa aa aa aa aa ribosome aa aa aa aa aa aa From gene to protein nucleus cytoplasm transcription translation DNA mRNA protein trait

  32. Transfer RNA structure • “Clover leaf” structure • anticodon on “clover leaf” end • amino acid attached on 3 end

  33. Oooh! Dehydration Synthesis again! Loading tRNA • Aminoacyl tRNA synthetase • enzyme which bonds amino acid to tRNA • bond requires energy • ATP  AMP • bond is unstable • so it can release amino acid at ribosome easily Trp C=O Trp Trp C=O H2O OH O OH C=O O activating enzyme tRNATrp A C C mRNA U G G anticodon tryptophan attached to tRNATrp tRNATrp binds to UGG condon of mRNA

  34. Ribosomes • Facilitate coupling of tRNA anticodon to mRNA codon • organelle or enzyme? • Structure • ribosomal RNA (rRNA) & proteins • 2 subunits • large • small E P A

  35. Ribosomes • A site (aminoacyl-tRNA site) • holds tRNA carrying next amino acid to be added to chain • P site (peptidyl-tRNA site) • holds tRNA carrying growing polypeptide chain • E site (exit site) • empty tRNA leaves ribosome from exit site Met C A U 5' G U A 3' E P A

  36. 3 2 1 Building a polypeptide • Initiation • brings together mRNA, ribosome subunits, initiator tRNA • Elongation • adding amino acids based on codon sequence • Termination • end codon release factor Leu Val Ser Met Met Ala Leu Met Met Leu Leu Trp tRNA C A G C G A C C C A A G A G C U A C C A U A U U A U G A A 5' 5' A A 5' C U U 5' A A G G A G U U G U C U U U G C A C U 3' G G U A A U A A C C mRNA 3' 3' 3' U G G U A A 3' E P A

  37. Asn Ala Val Ala Met Ala Cys How are the codons matched to amino acids? DNA 3 5 AUG-CGU-GUA-AAU-GCA-UGC-GCC 3 5 mRNA TAC-GCA-CAT-TTA-CGT-ACG-CGG “Anti” means “against”, so the anticodon must go “against” its complementary mRNA codon. So easy, a bird-brain like me can get it! mRNA codon aminoacid Met Ribosome tRNA anticodon http://vimeo.com/6812276 anti-codon AUG CGU CGG UAC UAC CAU GCA ACG UUA

  38. Destinations: • secretion • nucleus • mitochondria • chloroplasts • cell membrane • cytoplasm • etc… Protein targeting • Signal peptide • address label start of a secretory pathway

  39. RNA polymerase DNA Can you tell the story? aminoacids exon intron tRNA pre-mRNA 5' GTP cap http://www.biostudio.com/demo_freeman_protein_synthesis.htm mature mRNA aminoacyl tRNAsynthetase poly-A tail 3' large ribosomal subunit polypeptide http://www.youtube.com/watch?v=NJxobgkPEAo 5' tRNA small ribosomal subunit E P A ribosome

  40. The Central Dogma http://www.youtube.com/watch?v=J3HVVi2k2No

  41. Bacterial chromosome Protein Synthesis in Prokaryotes Transcription mRNA Psssst…no nucleus! Cell membrane Cell wall

  42. Prokaryotes DNA in cytoplasm circular chromosome naked DNA no introns Eukaryotes DNA in nucleus linear chromosomes DNA wound on histone proteins introns vs. exons intron = noncoding (inbetween) sequence exon = coding (expressed) sequence Prokaryote vs. Eukaryote genes intronscome out! eukaryotic DNA

  43. Translation in Prokaryotes • Transcription & translation are simultaneous in bacteria • DNA is in cytoplasm • no mRNA editing • ribosomesread mRNA as it is being transcribed

  44. Translation: prokaryotes vs. eukaryotes • Differences between prokaryotes & eukaryotes • time & physical separation between processes • takes eukaryote ~1 hour from DNA to protein • no RNA processing

  45. Any Questions?? What color would a smurf turnif he held his breath?

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