1. Chapter 17 From Gene to Protein

2. Question? u How does DNA control a cell? u By controlling Protein Synthesis. u Proteins are the link between genotype and phenotype.

3. Central Dogma DNA Transcription RNA Translation Polypeptide

5. Explanation u DNA - the Genetic code or genotype. u RNA - the message or instructions. u Polypeptide - the product for the phenotype.

6. DNA vs RNA DNA RNA Sugar – deoxyribose ribose Bases – ATGC AUGC Backbones – 2 1 Size – very large small Use – genetic code varied

7. Genetic Code u Sequence of DNA bases that describe which Amino Acid to place in what order in a polypeptide. u The genetic code gives the primary protein structure.

9. Genetic Code u Is based on triplets of bases. u Has redundancy; some AA's have more than 1 code. u Proof - make artificial RNA and see what AAs are used in protein synthesis (early 1960’s).

10. Codon u A 3-nucleotide “word” in the Genetic Code. u 64 possible codons known.

12. Codon Dictionary u Start- AUG (Met) u Stop- UAA UAG UGA u 60 codons for the other 19 AAs.

13. Code Redundancy u Wobble effect: Third base in a codon shows "wobble”. u First two bases are the most important in reading the code and giving the correct AA. The third base often doesn’t matter.

14. Code Evolution u The genetic code is nearly universal. u Ex: CCG = proline (all life) u Reason - The code must have evolved very early. Life on earth must share a common ancestor.

15. Reading Frame and Frame Shift u The “reading” of the code is every three bases (Reading Frame) u Ex: the red cat ate the rat u Frame shift – improper groupings of the bases u Ex: thr edc ata tet her at u The “words” only make sense if “read” in this grouping of three.

16. Transcription u Process of making RNA from a DNA template.

17. Transcription Steps 1. RNA Polymerase Binding 2. Initiation 3. Elongation 4. Termination

18. RNA Polymerase u Enzyme for building RNA from RNA nucleotides.

19. Binding u Requires that the enzyme find the “proper” place on the DNA to attach and start transcription.

20. Binding u Is a complicated process u Uses Promoter Regions on the DNA (upstream from the information for the protein) u Requires proteins called Transcription Factors.

22. TATA Box u Short segment of T,A,T,A u Located 25 nucleotides upstream from the initiation site. u Recognition site for transcription factors to bind to the DNA.

23. Transcription Factors u Proteins that bind to DNA before RNA Polymerase. u Recognizes TATA box, attaches, and “flags” the spot for RNA Polymerase.

24. Initiation u Actual unwinding of DNA to start RNA synthesis. u Requires Initiation Factors.

26. Elongation u RNA Polymerase untwists DNA 1 turn at a time and adds complimentary bases. u Exposes 10 DNA bases for pairing with RNA nucleotides.

28. Elongation u Enzyme moves 5’ 3’. u Rate is about 60 nucleotides per second.

29. Comment u Each gene can be read by sequential RNA Polymerases giving several copies of RNA. u Result - several copies of the protein can be made.

30. Termination u DNA sequence that tells RNA Polymerase to stop. u Ex: AATAAA u RNA Polymerase detaches from DNA after closing the helix.

32. Final Product u Pre-mRNA u This is a “raw” RNA that will need processing.

33. Modifications of RNA 1. 5’ Cap 2. Poly-A Tail 3. Splicing

34. 5' Cap u Modified Guanine nucleotide added to the 5' end. u Protects mRNA from digestive enzymes. u Recognition sign for ribosome attachment.

35. Poly-A Tail u 150-200 Adenine nucleotides added to the 3' tail u Protects mRNA from digestive enzymes. u Aids in mRNA transport from nucleus.

36. RNA Splicing u Removal of non-protein coding regions of RNA. u Coding regions are then spliced back together.

38. Introns u Intervening sequences. u Removed from RNA. u Some contain sequences that regulate gene expression and many affect gene products

39. Exons u Expressed sequences of RNA. u Translated into AAs.

40. Introns - Function u Left-over DNA (?) u Way to lengthen genetic message to protect coding regions. u Old virus inserts (?)

41. Introns- Function u Way to create new proteins with exon shuffling u New combinations of exons= new proteins for evolution

42. Final RNA Transcript

43. Translation u Process by which a cell interprets a genetic message and builds a polypeptide.

44. Materials Required u tRNA u Ribosomes u mRNA

45. Transfer RNA = tRNA u Made by transcription. u About 80 nucleotides long. u Carries AA for polypeptide synthesis.

46. Structure of tRNA You have a diagram of this u Has double stranded regions and 3 loops. u AA attachment site at the 3' end. u 1 loop serves as the Anticodon.

48. Anticodon u Region of tRNA that base pairs to mRNA codon. u Is a compliment to the mRNA bases, so reads the same as the DNA codon.

49. Example u DNA - GAC u mRNA - CUG u tRNA anticodon - GAC

50. Ribosomes u Two subunits made in the nucleolus. u Made of rRNA (60%)and protein (40%). u rRNA is the most abundant type of RNA in a cell.

51. Large subunit Proteins rRNA

52. Both sununits

53. Large Subunit u Has 3 sites for tRNA. u P site: Peptidyl-tRNA site - carries the growing polypeptide chain. u A site: Aminoacyl-tRNA site - holds the tRNA carrying the next AA to be added. u E site: Exit site

55. Translation Steps 1. Initiation 2. Elongation 3. Termination

56. Initiation u Brings together: u mRNA u A tRNA carrying the 1st AA u 2 subunits of the ribosome

57. Initiation Steps: 1. Small subunit binds to the mRNA. 2. Initiator tRNA (Met, AUG) binds to mRNA. 3. Large subunit binds to mRNA. Initiator tRNA is in the P-site

59. Initiation u Requires other proteins called "Initiation Factors”. u GTP used as energy source.

60. Elongation Steps: 1. Codon Recognition 2. Peptide Bond Formation 3. Translocation

61. Codon Recognition u tRNA anticodon matched to mRNA codon in the A site.

63. Peptide Bond Formation u A peptide bond is formed between the new AA and the polypeptide chain in the P-site. u Bond formation is by rRNA acting as a ribozyme

65. After bond formation u The polypeptide is now transferred from the tRNA in the P-site to the tRNA in the A-site.

67. Translocation u tRNA in P-site is released. u Ribosome advances 1 codon, 5’ 3’. u tRNA in A-site is now in the P-site. u Process repeats with the next codon.

69. Comment u Elongation takes 60 milliseconds for each AA added.

70. Termination u Triggered by stop codons. u Release factor binds in the A-site instead of a tRNA. u H 2 O is added instead of AA, freeing the polypeptide. u Ribosome separates.

72. Polyribosomes u Cluster of ribosomes all reading the same mRNA. u Another way to make multiple copies of a protein.

74. Comment u Polypeptide usually needs to be modified before it becomes functional.

75. Examples u Sugars, lipids, phosphate groups added. u Some AAs removed. u Protein may be cleaved. u Join polypeptides together (Quaternary Structure).

76. End of Part 1

77. Mutations u Changes in the genetic makeup of a cell. u May be at chromosome (review chapter 15) or DNA level

78. DNA or Point Mutations u Changes in one or a few nucleotides in the genetic code. u Effects - none to fatal.

79. Types of Point Mutations 1. Base-Pair Substitutions 2. Insertions 3. Deletions

80. Base-Pair Substitution u The replacement of 1 pair of nucleotides by another pair.

81. Sickle Cell Anemia

82. Types of Substitutions 1. Missense - altered codons, still code for AAs but not the right ones 2. Nonsense - changed codon becomes a stop codon.

85. Missense Effect u Can be none to fatal depending on where the AA was in the protein. u Ex: if in an active site - major effect. If in another part of the enzyme - no effect.

87. Nonsense Effect u Stops protein synthesis. u Leads to nonfunctional proteins unless the mutation was near the very end of the polypeptide.

88. Sense Mutations u The changing of a stop codon to a reading codon. u Result - longer polypeptides which may not be functional.

89. Insertions & Deletions u The addition or loss of a base in the DNA. u Cause frame shifts and extensive missense, nonsense or sense mutations.

91. Question? u Loss of 3 nucleotides is often not a problem. u Why? u Because the loss of a 3 bases or one codon restores the reading frame and the protein may still be able to function.

93. Mutagenesis u Process of causing mutations or changes in the DNA.

94. Mutagens u Materials that cause DNA changes. 1. Radiation ex: UV light, X-rays 2. Chemicals ex: 5-bromouracil

95. Spontaneous Mutations u Random errors during DNA replication.

96. Comment u Any material that can chemically bond to DNA, or is chemically similar to the nitrogen bases, will often be a very strong mutagen.

97. What is a gene? u A gene is a region of DNA that can be expressed to produce a final functional product. u The product can be a protein or a RNA molecule

98. Protein vs RNA u Protein – usually structure or enzyme for phenotype u RNA – often a regulatory molecule which will be discussed in future chapters.