Chapter 17 From Gene to Protein

1
Chapter 17From 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
1909 - Archibald Garrod

• Suggested genes control enzymes that catalyze
  chemical processes in cells.
• Inherited Diseases - inborn errors of
  metabolism where a person cant make an enzyme.

5
Example

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

6
George Beadle and Edward Tatum

• Worked with Neurospora and proved the link
  between genes and enzymes.

Neurospora Pink bread mold
7
Experiment

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

8
(No Transcript)
9
Results

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

10
Conclusion

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

11
Current Hypothesis

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

12
Central Dogma

• DNA
• Transcription
• RNA
• Translation
• Polypeptide

13
Explanation

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

14
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.

15
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.

16
If you use

• 2 bases 1 amino acid
• 42 16 amino acids
• Still not enough combinations.

17
If you use

• 3 bases 1AA
• 43 64 combinations
• More than enough for 20 amino acids.

18
(No Transcript)
19
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 1960s).

20
Codon

• A 3-nucleotide word in the Genetic Code.
• 64 possible codons known.

21
(No Transcript)
22
Codon Dictionary

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

23
For Testing

• Be able to read a DNA or RNA message and give
  the AA sequence.
• RNA Genetic Code Table will be provided.

24
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 doesnt matter.

25
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.

26
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.

27
Transcription

• Process of making RNA from a DNA template.

28
Transcription Steps

• 1. RNA Polymerase Binding
• 2. Initiation
• 3. Elongation
• 4. Termination

29
RNA Polymerase

• Enzyme for building RNA from RNA nucleotides.

30
Binding

• Requires that the enzyme find the proper place
  on the DNA to attach and start transcription.

31
Binding

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

32
(No Transcript)
33
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.

34
(No Transcript)
35
Transcription Factors

• Proteins that bind to DNA before RNA Polymerase.
• Recognizes TATA box, attaches, and flags the
  spot for RNA Polymerase.

36
(No Transcript)
37
Transcription Initiation Complex

• The complete assembly of transcription factors
  and RNA Polymerase bound to the promoter area of
  the DNA to be transcribed.

38
(No Transcript)
39
Initiation

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

40
(No Transcript)
41
Elongation

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

42
(No Transcript)
43
Elongation

• Enzyme moves 5 3.
• Rate is about 60 nucleotides per second.

44
(No Transcript)
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
Termination

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

47
(No Transcript)
48
Final Product

• Pre-mRNA
• 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

• 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
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.

53
RNA Splicing

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

54
(No Transcript)
55
Introns

• Intervening sequences.
• Removed from RNA.

56
Exons

• Expressed sequences of RNA.
• Translated into AAs.

57
Spliceosome

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

58
snRNA

• Small Nuclear RNA.
• 150 nucleotides long.
• Structural part of spliceosomes.

59
snRNPs

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

60
(No Transcript)
61
Result
62
Ribozymes

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

63
Introns - Function

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

64
Final RNA Transcript
65
Alternative Splicing

• The RNA can be spliced into different mRNAs.
• Each different mRNA produces a different
  polypeptide.
• Ex. variable regions of antibodies.

66
Another Example
67

• Bcl-XL inhibits apoptosis
• Bcl-XS induces apoptosis
• Two different and opposite effects!!

68
DSCAM Gene

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

69
Commentary

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

70
Translation

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

71
Materials Required

• tRNA
• Ribosomes
• mRNA

72
Transfer RNA tRNA

• Made by transcription.
• About 80 nucleotides long.
• Carries AA for polypeptide synthesis.

73
Structure of tRNA

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

74
(No Transcript)
75
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.

76
Example

• DNA - GAC
• mRNA - CUG
• tRNA anticodon - GAC

77
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.

78
Importance

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

79
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
(No Transcript)
81
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.

82
Large subunit
Proteins
rRNA
83
Both sununits
84
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
(No Transcript)
86
Translation Steps

• 1. Initiation
• 2. Elongation
• 3. Termination

87
Initiation

• Brings together
• mRNA
• A tRNA carrying the 1st AA
• 2 subunits of the ribosome

88
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
(No Transcript)
90
Initiation

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

91
Elongation Steps

• 1. Codon Recognition
• 2. Peptide Bond Formation
• 3. Translocation

92
Codon Recognition

• tRNA anticodon matched to mRNA codon in the A
  site.

93
(No Transcript)
94
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.

96
(No Transcript)
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.

98
(No Transcript)
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.

101
(No Transcript)
102
Polyribosomes

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

103
(No Transcript)
104
Prokaryotes
105
Homework

• Read Chapter 17
• Take home exam today
• Chapter 17 Fri. 1/25

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.

109
(No Transcript)
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 or DNA level

112
Chromosome Alterations

• Deletions
• Duplications
• Inversions
• Translocations

113
General Result

• Loss of genetic information.
• Position effects a gene's expression is
  influenced by its location to other genes.

114
(No Transcript)
115
Evidence of Translocation
Translocations
116
Cri Du Chat Syndrome

• Part of p arm of 5 has been deleted.
• Good survival.
• Severe mental retardation.
• Small sized heads common.

117
Philadelphia Chromosome

• An abnormal chromosome produced by a
  translocation of portions of chromosomes 9
  and 22.
• Causes chronic myeloid leukemia.

118
Mutation types - Cells

• Somatic cells or body cells not inherited
• Germ Cells or gametes - inherited

119
DNA or Point Mutations

• Changes in one or a few nucleotides in the
  genetic code.
• Effects - none to fatal.

120
Types of Point Mutations

• 1. Base-Pair Substitutions
• 2. Insertions
• 3. Deletions

121
(No Transcript)
122
Base-Pair Substitution

• The replacement of 1 pair of nucleotides by
  another pair.

123
Sickle Cell Anemia
124
Types of Substitutions

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

125
(No Transcript)
126
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.

127
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.

128
Nonsense Effect

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

129
Sense Mutations

• The changing of a stop codon to a reading codon.
• Result - longer polypeptides which may not be
  functional.
• Ex. heavy hemoglobin

130
Insertions Deletions

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

131
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.

132
Mutagenesis

• Process of causing mutations or changes in the
  DNA.

133
Mutagens

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

134
Spontaneous Mutations

• Random errors during DNA replication.

135
Comment

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

136
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.

137
Summary

• Be able to discuss RNA and protein processing.
• Be able to describe and discuss mutations.