Translating the Genetic Code

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Translating the Genetic Code

• Gene expression part 3

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An overview of gene expression
Figure 13.2
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The Idea of A Code

• 20 amino acids
• 4 nucleotides
• How do nucleic acids composed of 4 nucleotides
  specify the synthesis of proteins composed of 20
  different amino acids?

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The Coding Problem

• 1-1 correspondence NO 4 possibilities
• 2-1 correspondence NO 16 possibilities
• 3-1 correspondence YES 64 possibilities

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Determination of Triplet Nature of Code

• Aacridines flavinoids cause single nucleotide
  deletions and insertions respectively
• Insertion of 1 base shifts frame creates
  non-sense mutation
• Deletion of base in mutant restores frame
  (suppresses 1st mutation)
• Recombination between mutants showed that
  insertion of 1 or 2 bases or removal of 1 or 2
  bases doesn't restore frame, but insertion of 3
  or removal of 3 allows frame to continue

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Cricks Experiments to Determine Triplet Nature
of Code
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So.. The code is 3 letter words, but what about
punctuation?
GROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW
a b c

• There is a message, but one must start at the
  right place to read it
• Code written in three letter words - codon
• There are three reading frames, but only one
  gives an intelligible message frame b
• NOW THE CAT SAW THE DOG BUT DID NOT RUN
• A start codon (NOW) and a stop codon (END) define
  the frame to use

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Any frame is potentially the reading frame!
ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW
a b c
OWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW
a b c
The actual reading frame is called the Open
Reading Frame or ORF
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Reading Frames Mutation Types

• Frame shift mutations
• Original reading frame is frame a
• Insertions or deletions shift the reading frame

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Reading Frames Mutations
ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW
a b c

• Once a ribosome begins translation in a
  particular frame (a) it does not shift frames
• Therefore, if a mutation shifts the reading frame
  in the mRNA, the ribosome will read the wrong
  frame.

NOW THE CAT SAW THE ADO GBU TDI DNO TRU NEN DSE
W..
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Deciphering the Code

• Each amino acid in a protein is specified by 3
  nucleotides of codon
• Each codon specifies only ONE amimo acid
• There are 64 possible codons but only 20 amino
  acids
• Degeneracy
• An amino acid can be specified by multiple codons
• A given codon still only specifies only one amino
  acid

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Deciphering the Code Three Approaches

• Synthesis and translation of homopolymer RNA
  ratio polymer RNA followed by amino acid analysis
• Synthesis and translation of di, tri and tetra
  nucleotide repetitive RNA polymers followed by
  amino acid analysis
• Triplet RNA-tRNA binding assay and amino acid
  analysis

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Synthetic RNA Templates and In Vitro Translation

• Synthesis of homopolymeric RNAs
• UUUUUUUUUU, AAAAAAAAAA, CCCCCCCCC, GGGGGGGGG
• When translated produced polypeptides
• poly phenylalanine (UUU), polyproline (CCC),
  polylysine (AAA), and polyglycine (GGG)
• Therefore 4 codons were determined
• UUU phe
• CCC pro
• AAA lys
• GGG gly

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Decoding the Genetic Code

• The enzyme polynucleotide phosphorylase
• polymerizes ribonucleoside diphosphates (NDPs)
  into RNA
• It does not use a template, the order of
  nucleotides is random
• The nucleotide sequence is controlled by the
  relative abundance of NDPs
• For example, if 70 GDP and 30 UDP are mixed
  together, then

Radiolabeled Amino Acid Added () Amino Acid Incorporated
Glycine (GGG GGU, UGG, GUG) 49 (34 15)
Valine(GGU, UGG, GUG, GUU, UUG, UGU) 21(15 6)
Tryptophan(UGG, GUG, GGU) 15
Cysteine(UUG, GUU, UGU) 6
Leucine (UUG, GUU, UGU) 6
Phenylalanine (UUU) 3
Possible Codons Percentage in the Random Polymer
GGG 0.7 x 0.7 x 0.7 0.34 34
GGU 0.7 x 0.7 x 0.3 0.15 15
GUU 0.7 x 0.3 x 0.3 0.06 6
UUU 0.3 x 0.3 x 0.3 0.03 3
UGG 0.3 x 0.7 x 0.7 0.15 15
UUG 0.3 x 0.3 x 0.7 0.06 6
UGU 0.3 x 0.7 x 0.3 0.06 6
GUG 0.7 x 0.3 x 0.7 0.15 15
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Polymers of Nucleotide Repeats
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Trinucleotide-tRNA Binding Analysis
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(No Transcript)
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• Special codons
• AUG (which specifies methionine) start codon
• AUG specifies internal methionines also
• UAA, UAG and UGA termination, or stop, codons
• The code is degenerate
• More than one codon can specify the same amino
  acid
• For example GGU, GGC, GGA and GGG all code for
  lysine
• In most instances, the third base is the
  degenerate base
• It is sometime referred to as the wobble base
• The code is nearly universal
• Only a few rare exceptions have been noted