1. Chapter 15 From Genes to Proteins

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

3. For tests: u Name(s) of experimenters u Outline of the experiment u Result of the experiment and its importance

4. 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 (because of 4 th 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. 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.

15. 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.

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

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

19. 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).

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

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

23. 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.

24. 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.

25. 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.

26. 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 tat her at u The “words” only make sense if “read” in this grouping of three.

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

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

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

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

31. 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.

33. 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.

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

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

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

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

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

45. 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.

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

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

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

50. 5' Cap u Modified Guanine nucleotide added to the 5' end. u Protects mRNA from digestive enzymes. u Recognition sign for ribosome attachment.

51. 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.

52. 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.

53. u Let’s see Transcription in motion… u http://www.hhmi.org/biointera ctive/media/DNAi_transcriptio n_vo2-lg.mov http://www.hhmi.org/biointera ctive/media/DNAi_transcriptio n_vo2-lg.mov

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

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

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

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

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

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

62. Result

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

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

65. Final RNA Transcript

66. Alternative Splicing u The RNA can be spliced into different mRNA’s. u Each different mRNA produces a different polypeptide. u Ex. – variable regions of antibodies.

67. Another Example

68. u Bcl-X L – inhibits apoptosis u Bcl-X S – induces apoptosis u Two different and opposite effects!!

69. DSCAM Gene u Found in fruit flies u Has 100 potential splicing sites. u Could produce 38,000 different polypeptides u Many of these polypeptides have been found

70. Commentary u Alternative Splicing is going to be a BIG topic in Biology. u About 60% of genes are estimated to have alternative splicing sites. u One gene does not equal one polypeptide.

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

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

89. 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

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

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

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

95. 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

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

98. 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.

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

101. 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.

103. u Let’s see Translation in motion… u http://www.hhmi.org/biointera ctive/media/DNAi_translation _vo2-lg.mov http://www.hhmi.org/biointera ctive/media/DNAi_translation _vo2-lg.mov

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 May be at chromosome or DNA level

113. Chromosome Alterations u Deletions u Duplications u Inversions u Translocations

114. General Result u Loss of genetic information. u Position effects: a gene's expression is influenced by its location to other genes.

116. Evidence of Translocation Translocations

117. Cri Du Chat Syndrome u Part of p arm of #5 has been deleted. u Good survival. u Severe mental retardation. u Small sized heads common.

118. Philadelphia Chromosome u An abnormal chromosome produced by a translocation of portions of chromosomes 9 and 22. u Causes chronic myeloid leukemia.

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

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

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

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

124. Sickle Cell Anemia

125. u Lets see how this mutation will affect the cell… u http://www.hhmi.org/biointera ctive/media/DNAi_sicklecell- lg.mov http://www.hhmi.org/biointera ctive/media/DNAi_sicklecell- lg.mov

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

128. 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”.

129. 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.

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

131. 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

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

133. 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.

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

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

136. Spontaneous Mutations u Random errors during DNA replication.

137. 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.

138. 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.

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