1. Chapter 17 From Gene to Protein

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

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

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

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

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

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

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

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

13. Current Hypothesis
One Gene - One Polypeptide Hypothesis (because of 4th degree structure).

14. DNA Transcription RNA Translation Polypeptide
Central Dogma
DNA Transcription RNA Translation Polypeptide

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

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

18. Code Basis If you use: 1 base = 1 amino acid 4 bases = 4 amino acids
41 = 4 combinations, which are not enough for 20 AAs.

19. If you use: 2 bases = 1 amino acid Ex – AT, TA, CA, GC
42 = 16 amino acids
Still not enough combinations.

20. If you use: 3 bases = 1AA Ex – CAT, AGC, TTT 43 = 64 combinations
More than enough for 20 amino acids.

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

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

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

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

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

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

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

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

31. Transcription
Process of making RNA from a DNA template.

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

33. RNA Polymerase
Enzyme for building RNA from RNA nucleotides.

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

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

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

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

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

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

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

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

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

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

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

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

54. 5' Cap Modified Guanine nucleotide added to the 5' end.
Protects mRNA from digestive enzymes.
Recognition sign for ribosome attachment.

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

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

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

59. Introns
Intervening sequences.
Removed from RNA.

60. Exons
Expressed sequences of RNA.
Translated into AAs.

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

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

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

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

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

68. Final RNA Transcript

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

70. Materials Required
tRNA
Ribosomes
mRNA

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

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

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

75. Example
DNA - GAC
mRNA - CUG
tRNA anticodon - GAC

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

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

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

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

81. Large subunit
Proteins
rRNA

82. Both sununits

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

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

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

87. 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

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

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

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

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

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

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

99. Comment
Elongation takes 60 milliseconds for each AA added.

100. Termination Triggered by stop codons.
Release factor binds in the A-site instead of a tRNA.
H2O is added instead of AA, freeing the polypeptide.
Ribosome separates.

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

104. Prokaryotes

105. Assignments Read Chapter 17, Chapter 9 in Hillis Chapter 17 – Mon.
Lab Summary – Wed. (skip # 4, 9)

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

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

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

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

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

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

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

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

115. Sickle Cell Anemia

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

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

119. Comment
Silent mutations may still have an effect by slowing down the “speed” of making the protein.
Reason – harder to find some tRNAs than others.

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

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

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

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

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

129. Mutagenesis
Process of causing mutations or changes in the DNA.

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

131. Spontaneous Mutations
Random errors during DNA replication.

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

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

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

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

136. Summary Be able to discuss RNA and protein processing.
Be able to describe and discuss mutations.
Be able to discuss “what is a gene”.