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. 1909 - Archibald Garrod u Suggested genes control enzymes that catalyze chemical processes in cells. u Inherited Diseases - “inborn errors of metabolism” where a person can’t make an enzyme.

5. Example u Alkaptonuria - where urine turns black after exposure to air. u Lacks - an enzyme to metabolize alkapton.

6. George Beadle and Edward Tatum u Worked with Neurospora and proved the link between genes and enzymes. Neurospora Pink bread mold

7. Experiment u Grew Neurospora on agar. u Varied the nutrients. u Looked for mutants that failed to grow on minimum agar.

9. Results u Three classes of mutants for Arginine Synthesis. u Each mutant had a different block in the Arginine Synthesis pathway.

10. Conclusion u Mutations were abnormal genes. u Each gene dictated the synthesis of one enzyme. u One Gene - One Enzyme Hypothesis.

11. Current Hypothesis u One Gene - One Polypeptide Hypothesis. u We now know proteins may have 4th degree structure.

12. Central Dogma DNA Transcription RNA Translation Polypeptide

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

14. DNA STRUCTURE; REPLICATION ANIMATION STEPS u DNA makes DNA.mht DNA makes DNA.mht

16. 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.

17. "for their interpretation of the genetic code and its function in protein synthesis" The Nobel Prize in Physiology or Medicine 1968 Har Gobind Khorana Robert W. Holley Marshall W. Nirenberg 1922-19931922-1927-

18. Code Basis If you use: u 1 base = 1 amino acid u 4 bases = 4 amino acids u 4 1 = 4 combinations, which are not enough for 20 AAs.

19. If you use: u 2 bases = 1 amino acid u 4 2 = 16 amino acids u Still not enough combinations.

20. If you use: u 3 bases = 1AA u 4 3 = 64 combinations u More than enough for 20 amino acids.

22. 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).

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

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

26. For Testing: u Be able to “read” a DNA or RNA message and give the AA sequence. u RNA Genetic Code Table will be provided.

27. Code Redundancy u 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.

28. Reading Frame u The “reading” of the code is every three bases. u Ex: the red cat ate the rat u Ex: ATT GAT TAC ATT u The “words” only make sense if “read” in this grouping of three.

29. 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.

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

31. Movie - preview

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

33. RNA Polymerase u Enzyme for building RNA from RNA nucleotides. u Prokaryotes - 1 type u Eukaroyotes- 3 types

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

35. RNA Polymerase Binding Needs: u Promoter Regions on the DNA. u Transcription Factors.

36. Promoters u Regions of DNA where RNA Polymerases can bind. u It’s where transcription is initiated. u About 100 nucleotides long. Include initiation site and recognition areas for RNA Polymerase.

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

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

42. Transcription Initiation Complex u The complete assembly of transcription factors and RNA Polymerase bound to the promoter area of the DNA to be transcribed.

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

46. Elongation u RNA Polymerase untwists DNA 1 turn at a time. u Exposes 10 DNA bases for pairing with RNA nucleotides.

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

50. 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. Efficiency

51. Termination u DNA sequence that tells RNA Polymerase to stop. u Most common in eukaryotes: AATAAA u RNA Polymerase detaches from DNA after closing the helix.

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

54. Modifications of RNA (Splicing & Dicing) 1. 5’ Cap 2. Poly-A Tail 3. Splicing

55. 5' Cap u Modified Guanine nucleotide added to the 5' end. u Protects mRNA from digestive enzymes. u Recognition sign for ribosome attachment.

56. 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.

57. Comment u The head and tail areas often contain “leaders” and “trailers”, areas of RNA that are not read. u Similar to leaders or trailers on cassette tapes.

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

60. Introns u Intervening sequences. u Removed from RNA.

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

62. Spliceosome u Cut out Introns and join Exons together. u Made of snRNA and snRNP.

63. snRNPs u ("snurps") u Small Nuclear Ribonucleoprotiens u Made of snRNA and proteins. u Join with other proteins to form a spliceosome.

64. snRNA u Small Nuclear RNA. u 150 nucleotides long. u Structural part of spliceosomes.

66. Result

67. Summary of RNA processing u In eukaryotes, RNA polymerase produces a “primary transcript”, an exact RNA copy of the gene. u A cap is put on the 5’ end. u The RNA is terminated and poly-A is added to the 3’ end. u All introns are spliced out. u At this point, the RNA can be called messenger RNA. It is then transported out of the nucleus into the cytoplasm, where it is translated.

68. Ribozymes u RNA molecules that act as enzymes. u Are sometimes Intron RNA and cause splicing without a spliceosome.

69. Introns - Function u Left-over DNA (?) u Way to lengthen genetic message. u Old virus inserts (?) u Way to create new proteins.

70. Final RNA Transcript

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

72. Materials Required u tRNA u Ribosomes u mRNA

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

74. Structure of tRNA u Has double stranded regions and 3 loops. u AA attachment site at the 3' end. u 1 loop serves as the Anticodon.

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

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

78. Comment u "Wobble" effect allows for 45 types of tRNA instead of 61. u Reason - in the third position, U can pair with A or G. u Inosine (I), a modified base in the third position can pair with U, C, or A.

79. Importance u Allows for fewer types of tRNA. u Allows some mistakes to code for the same AA which gives exactly the same polypeptide.

80. Aminoacyl-tRNA Synthetases u Family of Enzymes. u Add AAs to tRNAs. u Active site fits 1AA and 1 type of tRNA. u Uses a “secondary genetic” code to load the correct AA to each tRNA.

82. 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.

83. Large subunit Proteins rRNA

84. Both sununits

85. 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

87. Movie - preview

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

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

90. 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

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

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

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

96. 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

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

99. 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.

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

102. 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.

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

106. Prokaryotes

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

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

109. Signal Hypothesis u “Clue” on the growing polypeptide that causes ribosome to attach to ER. u All ribosomes are “free” ribosomes unless clued by the polypeptide to attach to the ER.

111. Result u Protein is made directly into the ER. u Protein targeted to desired location (e.g. secreted protein). u “Clue” (the first 20 AAs are removed by processing).

112. Mutations u Changes in the genetic makeup of a cell. u Chapter 15 covered large- scale chromosomal mutations. (hint - review these)

113. Mutation types - Cells u Somatic cells or body cells – not inherited u Germ Cells or gametes - inherited

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

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

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

118. Sickle Cell Anemia

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

121. Question? u What will the "Wobble" Effect have on Missense? u If the 3rd base is changed, the AA may still be the same and the mutation is “silent”.

122. 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.

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

124. Sense Mutations u The changing of a stop codon to a reading codon. u Result - longer polypeptides which may not be functional. u Ex. “heavy” hemoglobin

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

126. Frame Shift u The “reading” of the code is every three bases. u Ex: the red cat ate the rat u Ex: thr edc ata tat her at u The “words” only make sense if “read” in this grouping of three.

127. 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.

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

129. Spontaneous Mutations u Random errors during DNA replication.

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

131. 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.

132. The Ames Test u Measures the mutagenic strength of various chemicals. u Looks for back-mutations in bacteria.

133. Ames Results u The more back mutations, the more colonies appear, the stronger the mutagenic effect of the material. u Usually compared to + and - controls.

134. Summary u Know Beadle and Tatum. u Know the central dogma. u Be able to “read” the genetic code. u Be able to describe the events of transcription and translation.

135. Summary u Be able to discuss RNA and protein processing. u Be able to describe and discuss DNA mutations.