From Gene to Protein

1. 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? RNA DNA protein trait DNA gets all the glory, but proteins do all the work!

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. X rays or ultraviolet light Wild-type Neurospora asexual spores Minimal medium Growth on complete medium spores Select one of the spores Grow on complete medium Test on minimal medium to confirm presence of mutation Minimal media supplemented only with… Choline Pyridoxine Riboflavin Minimal control Nucleic acid Arginine Niacin Inositol Folic acid p-Amino benzoic acid Thiamine Beadle & Tatum create mutations positive control negative control mutation identified experimentals amino acidsupplements

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

8. Transcription fromDNA nucleic acid languagetoRNA nucleic acid language

9. RNA • ribose sugar • N-bases • Adenine • Guanine • Cytosine • Uracil instead of Thymine • lots of RNAs • mRNA, tRNA, rRNA, siRNA… transcription DNA RNA

10. Transcription • Making mRNA • transcribed DNA strand = Template • untranscribed DNA strand = Coding • 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 • binding site before beginning of gene • TATA box • binding site for RNA polymerase & transcription factors • Regulator • binding site far upstream of gene • turns transcription on HIGH

13. Transcription Factors • Initiation complex • Transcription Factors • 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 = uncoded DNA • inbetween sequence eukaryotic DNA

16. intron = noncoding (inbetween) sequence exon = coding (expressed) sequence mRNA splicing • Post-transcriptional processing • eukaryotic mRNA needs work after transcription • 5’ cap is added • Spliceosome • edit out introns • Poly A Tail ~10,000 bases eukaryotic DNA pre-mRNA primary mRNA transcript ~1,000 bases mature mRNA transcript spliced mRNA

17. Splicing must be accurate • No room for mistakes! • a single base added or lost throws off the Reading frame AUGCGGCUAUGGGUCCGAUAAGGGCCAU AUGCGGUCCGAUAAGGGCCAU AUG|CGG|UCC|GAU|AAG|GGC|CAU Met|Arg|Ser|Asp|Lys|Gly|His AUGCGGCUAUGGGUCCGAUAAGGGCCAU AUGCGGGUCCGAUAAGGGCCAU AUG|CGG|GUC|CGA|UAA|GGG|CCA|U Met|Arg|Val|Arg|STOP|

18. snRNPs snRNA intron exon exon 5' 3' spliceosome 5' 3' lariat 5' 3' exon exon mature mRNA excised intron 5' 3' RNA splicing enzymes • snRNP • snRNA • proteins • Spliceosome • several snRNPs • recognize splice site sequence • cut & paste gene

19. Most introns start from the sequence GU and end with the sequence AG (in the 5' to 3' direction).  • Splice donor and Splice acceptor • Another important sequence is called the branch site located 20 - 50 bases upstream of the acceptor site.   The consensus sequence of the branch site is "CU(A/G)A(C/U)", where A is conserved in all genes.

20. Example of splice sites. • In over 60% of cases, the exon sequence is (A/C)AG at the donor site, and G at the acceptor site.

21. Alternative splicing • A single gene may code for more than one protein • when is an intron not an intron… • different segments treated as exons

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 • 5’ cap • 3’ poly A Tail

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. Translation fromnucleic acid languagetoamino acid language

25. 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

26. TACGCACATTTACGTACGCGG DNA AUGCGUGUAAAUGCAUGCGCC mRNA AUGCGUGUAAAUGCAUGCGCC mRNA MetArgValAsnAlaCysAla protein ? mRNA codes for proteins in triplets codon

27. 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

28. 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” • Start • AUG • methionine • Stop • UGA, UAA, UAG

29. GCA UAC CAU Met Arg Val How are the codons matched to amino acids? 3 5 TACGCACATTTACGTACGCGG DNA 5 3 AUGCGUGUAAAUGCAUGCGCC mRNA codon 3 5 tRNA anti-codon aminoacid

30. Inosine is a nucleoside that is formed when hypoxanthine is attached to a ribose ring via a β-N9-glycosidic bond and is a precursor to adenine. • Inosine is commonly found in tRNAs and is essential for proper translation of the genetic code in wobble base pairs. Inosine found in tRNA

31. Degenerate code Degeneracy of the genetic code: one amino acid may be specified by more than 1 codon Example: Alanine is encoded by: GCU GCC GCA GCG 2nd position 3rd (Wobble) position 1st position

32. The Wobble Hypothesis 3 2 1 Anticodon (3’) G-C-I (5’) Codon (5’) C-G-A (3’) 1 2 3 There are four statements that form the basic Wobble hypothesis: 1. The first 2 bases of the mRNA are the major determinants and form strong base pairs with the tRNA, while the third position is wobbly. 3 2 1 Anticodon (3’) G-C-I (5’) Codon (5’) C-G-U (3’) 1 2 3 3 2 1 Anticodon (3’) G-C-I (5’) Codon (5’) C-G-C (3’) 1 2 3

33. (3’) X-Y-A (5’) (5’) Y-X-U (3’) (3’) X-Y-C (5’) (5’) Y-X-G (3’) (3’) X-Y-U(5’) (5’) Y-X-A (3’) (3’) X-Y-G (5’) (5’) Y-X-C (3’) (3’) X-Y-U (5’) (5’) Y-X-G (3’) (3’) X-Y-G(5’) (5’) Y-X-U(3’) (3’) X-Y- I (5’) (5’) Y-X-U (3’) (3’) X-Y- I(5’) (5’) Y-X-A (3’) (3’) X-Y- I(5’) (5’) Y-X-C (3’) The Wobble Hypothesis anticodon codon (3’) X-Y-N (5’) (5’) Y-X-N (3’) 2. The 1st base of the anticodon pairs with the 3rd base of the codon. • If the 1st base of the anticodon is an A or C, then one codon is recognized. • If the 1st base of the anticodon is a U or G, then two codons are recognized. • If the 1st base of the anticodon is an I, then three codons are recognized

34. Leu Ser UUA CUA AGU UCU (3’) A-A-U (5’) (5’) U-U-A (3’) (3’) G-A-U (5’) (5’) C-U-A (3’) (3’) A-A-U (5’) (5’) A-G-U (3’) (3’) A-G-A (5’) (5’) U-C-U (3’) The Wobble Hypothesis 3. If an amino acid is specified by several different codons, then the codons that differ in either of the first 2 bases require different tRNAs

35. The Wobble Hypothesis 4. A minimum of 32 tRNAs is required to translate all 61 codons X is either U, A, G, C Y is either U, A, G, C X-Y-G (16 combinations) X-Y-I (16 combinations) I = inosine (forms hydrogen bonds with C, U, or A)

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

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

38. 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 Aminoacyl-tRNA synthetase O OH C=O O tRNATrp A C C mRNA U G G anticodon tryptophan attached to tRNATrp tRNATrp binds to UGG condon of mRNA

39. Ribosomes • Facilitate coupling of tRNA anticodon to mRNA codon • organelle or enzyme? • Structure • Large and small subunit • Made of • Mainly RNA • Protein E P A

40. 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

41. 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

42. Destinations: • secretion • nucleus • mitochondria • chloroplasts • cell membrane • cytoplasm • etc… Protein targeting • Specific Locations • Signal peptide and SRP • Translocation complex start of a secretory pathway

43. Can you tell the story?

44. 20-30b 5' 3' 5' 3' The Transcriptional unit enhancer exons 1000+b transcriptional unit 3' 5' TAC ACT DNA introns

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

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

47. Translation: prokaryotes vs. eukaryotes • Differences between prokaryotes & eukaryotes • time & physical separation between processes • takes eukaryote ~1 hour from DNA to protein • RNA processing

48. Prokaryotes ________________ ________________ ________________ ________________ ________________ Eukaryotes ________________ ________________ ________________ ________________ ________________ intron = noncoding (inbetween) sequence exon = coding (expressed) sequence Prokaryote vs. Eukaryote genes eukaryotic DNA

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