1. Chapter 17: From Gene to Protein (Protein Synthesis)

2. Essential Knowledge
3.a.1 – DNA, and in some cases RNA, is the primary source of heritable information ( ).
3.c.1 – Changes in genotype can result in changes in phenotype (17.5).

3. Question? How does DNA control a cell? (or identify a phenotype)
By controlling protein synthesis (otherwise known as gene expression)
Proteins are the link between genotype and phenotype

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
Symptoms reflect person’s inability to make proteins/enzymes

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

6. George Beadle and Edward Tatum
Worked with Neurospora and proved the link between genes and enzymes
Grew Neurospora on agar
Varied the nutrients in the agar
Looked for mutants that failed to grow on minimum agar

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

9. Current Hypothesis One Gene - One Polypeptide Hypothesis.
Why change? Not all proteins are enzymes
We now know proteins may have 4th degree structure.

10. Central Dogma Transcription Translation Polypeptide chain
DNA
Transcription
RNA
Translation
Polypeptide chain
(will become protein)

11. Explanation DNA – the genetic code or genotype
RNA - the message or instructions
Polypeptide - the end product for the phenotype

12. Why is there an RNA intermediate?
Evolutionary adaptation:
Check-point in process
Provides protection for DNA code
More copies can be made simultaneously

13. Genetic Code
Sequence of DNA bases that describe which amino acid to place in what order in a polypeptide chain
The genetic code gives ONLY the primary protein structure
All other protein structures result from chemical interactions amongst primary protein structure

15. Genetic Code Is based on triplets of bases (called codons)
Has redundancy; some AA's have more than 1 code/3-base codon
Proof - make artificial RNA and see what AAs are used in protein synthesis (early 1960’s)

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

17. Codon Amino acid

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

19. Code Redundancy Third base in a codon shows "wobble” effect
First two bases are the most important in reading the code and giving the correct AA
The third base often doesn’t matter
This allows for mistakes during DNA replication

20. Reading Frame The “reading” of the code is every three bases
Ex: the red cat ate the rat
Ex: ATT GAT TAC ATT
The “words” (codons) only make sense if “read” in this grouping of three (in correct “letter” order)

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

22. Protein Synthesis Intro
Intro movie

23. Protein Synthesis Intro
Step 1: Transcription:
DNA  mRNA
Step 2: Translation:
mRNA  tRNA  Am. Acid  Polypep. chain
Polypeptide chain then becomes protein

24. Transcription Process of making RNA from a DNA template
RNA type: mRNA (messenger)
Intermediate type
Takes place in nucleus (in eukaryotes)

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

26. RNA Polymerase Enzyme for building RNA from RNA nucleotides
Prokaryotes - 1 type
Eukaroyotes- 3 types
Splits two DNA strands apart
Hooks RNA nucleotides together (as they pair with DNA)

27. 1st Step: RNA Polymerase Binding
Requires that the enzyme find the “proper” place on the DNA to attach and start transcription
Different from DNA polymerase
Doesn’t require an RNA primer

28. RNA Polymerase Binding Needs:
Promoter Regions (on the DNA)
Special sequences of DNA nucleotides that “tell” cell where transcription begins
Transcription Factors
Proteins

29. Promoters Regions of DNA where RNA Polymerases can bind
About 100 nucleotides long. Include initiation site and recognition areas for RNA Polymerase
Also “decide” which DNA strand to use

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

34. Transcription Factors
Proteins that bind to DNA before RNA Polymerase
Recognizes TATA box, attaches, and “flags” the spot for RNA Polymerase
RNA poly won’t attach unless these are present

36. Transcription Initiation Complex
The complete assembly of
1) transcription factors and
2) RNA Polymerase
Bound to the promoter area of the DNA to be transcribed

38. 2nd Step: Initiation 2nd step of transcription
Actual unwinding of DNA to start RNA synthesis.
Requires Initiation Factors

40. Comment Getting Transcription started is complicated
Gives many ways to control which genes are decoded and which proteins are synthesized

41. 3rd Step: Elongation 3rd step in transcription
RNA Polymerase untwists DNA 1 turn at a time
Exposes 10 DNA bases for pairing with RNA nucleotides

43. Elongation Adds nucleotides to 3` end of growing RNA strand
Enzyme moves 5`  3` (of RNA strand)
Rate is about 60 nucleotides per second

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

46. 4th Step: Termination DNA sequence that tells RNA Polymerase to stop
Ex: AATAAA
RNA Polymerase detaches from DNA after closing the helix

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

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

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

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

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

54. Introns and Exons Introns: Exons: Intervening sequences
Removed from RNA.
Exons:
Expressed sequences of RNA
Translated into AAs

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

56. Translation Poster Requirements
1. What is translation? (definition)
2. What is needed?
3. Specifics & Structure of tRNA
4. Where does it occur?
5. Ribosome specifics – be sure to include the specifics of each subunit
6. Steps of translation & details of each step
7. What bonds are formed?
8. Illustration

57. 2nd step of Protein Synthesis: Translation
Process by which a cell interprets a genetic message and builds a polypeptide
Location: mRNA moves from nucleus to cytoplasm and ribosomes

58. Materials Required for translation
tRNA
Ribosomes
mRNA

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

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

62. 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
Example:
DNA- GAC
mRNA – CUG
tRNA anticodon - GAC

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

64. 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

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

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

68. 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

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

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

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

73. 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
After bond formation:
The polypeptide is now transferred from the tRNA in the P-site to the tRNA in the A-site

74. 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

76. 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

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

80. Prokaryotes: Prok. vs. Euk. Protein Synthesis Video

81. Polypeptide vs. Protein
Polypeptide usually needs to be modified before it becomes functional
Ex:
Sugars, lipids, phosphate groups added
Some AAs removed
Protein may be cleaved
Join polypeptides together (Quaternary Structure)

82. Mutations Changes in the genetic make-up of a cell
Chapter 15 covered large-scale chromosomal mutations
(Hint - review these!)

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

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

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

87. Base-Pair Substitution
The replacement of 1 pair of nucleotides by another pair
Ex: Sickle cell anemia

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

91. 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”

92. 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

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

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

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

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

98. 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

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

102. Chernobyl video

103. Summary
Recognize the relationship between genes and enzymes (proteins) as demonstrated by the experiments of Beadle and Tatum.
Identify the flow of genetic information from DNA to RNA to polypeptide (the “Central Dogma”).
Read DNA or RNA messages using the genetic code.
Recognize the steps and procedures in transcription.

104. Summary Identify methods of RNA modification.
Recognize the steps and procedures in translation.
Recognize categories and consequences of base-pair mutations.
Identify causes of mutations.
Be able to recognize and discuss “What is a gene?”