Genetic Code 1
Presented with Respect to
Prof. AMRITPAL SINGH
Presented by
Patel Hiren M.
M.V.Sc. (Anim.Biotechnology)
INTRODUCTION
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The genetic code consists of 64 triplets of nucleotides
called as Codons
Each codon encodes for one of the 20 amino acids
Most of the amino acids being encoded by more than one
codon (redundancy)
The genetic code can be expressed as either RNA codons
or DNA codons
Pathway for Gene Expression
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How do Nucleotides Specify 20 Amino Acids?
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4 different nucleotides (A, G, C, U)
20 amino acids
Possible codes:
1 letter code 4 AAs <20
2 letter code 4 x 4 = 16 AAs <20
3 letter code 4 x 4 x 4 = 64 AAs >>20
A triplet code is the most efficient way to code for all 20
amino acids, Shown by Crick et al in 1961
Evidence that the genetic code is a triplet code
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1960s: Francis Crick et al.
Studied frameshift mutations in bacteriophage T4 (& E. coli),
induced by the mutagen proflavin.
Proflavin causes the addition or deletion of a base pair in the
DNA.
Two ways to identify mutant T4:
Growth with E. coli B:
r+(wild type) Turbid plaques
rII (mutant) Clear plaques
Growth with E. coli K12 ( ):
r+ (wild type) Growth
rII (mutant) No growth
1. Discovered that frame shift mutations (addition or deletion) resulted in a
different sequence of amino acids.
Fig. 6.5
20. Also discovered that r+ mutants treated with proflavin could be restored to
the wild type (revertants).
ü deletion (-) corrects addition (+) or vice versa
4. Combination of three r+ mutants routinely yielded revertants, unlike
other multiple combinations.
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Fig. shows how three nearby + (addition) mutations restore the reading
frame, giving normal or near-normal function.
How was the Genetic Code Deciphered?
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1. Cell-free, protein synthesizing machinery isolated from E. coli.
(ribosomes, tRNAs, protein factors, radio-labeled amino acids).
Synthetic mRNA containing only one type of base:
UUU = Phe, CCC = Pro, AAA = Lys, GGG = ? (unstable)
7. Synthetic copolymers (CCC, CCA, CAC, ACC, CAA, ACA,
AAC, AAA) composed of two different bases:
Pro, Lys (already defined) + Asp, Glu, His, & Thr
Proportion (%AC) varied to determine exactly which codon specified
which amino acid.
9. Synthetic polynucleotide of known composition:
UCU CUC UCU CUC Ser Leu Ser Leu
4. Ribosome binding assays of Nirenberg and Leder (1964) (ribosomes,
tRNAs charged w/AAs, RNA trinucleotides).
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ü Protein synthesis does not occur.
ü Only one type of charged tRNA will bind to the tri - nucleotide.
mRNA UUU codon
tRNA AAA (with Phe) anti-codon
mRNA UCU codon
tRNA AGU (with Ser) anti-codon
mRNA CUC codon
tRNA GAG (with Leu) anti-codon
ü Identified 50 codons using this method.
16. Combination of different methods eventually identified 61 codons, the
other 3 do not specify amino acids (stop-codons).
Triplet Binding Technique
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1. Ability to make 3 base length RNA’s in specific
sequence
2. 3 base length RNA could bind to ribosome
3. Synthesized short RNA’s and added them to system
containing ribosomes, tRNA’s, amino acids, etc.
4. Nitrocellulose filter paper: key ingredient because
ribosomes adhere to paper
Triplet Binding Technique
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5. Mix RNA, ribosomes , etc., and pour through filter
paper
6. Ribosome + mRNA + tRNA (with amino acid) that is
complementary to mRNA sticks to filter paper
7. Serial experiment: label 1 amino acid each time
8. Assay filter paper for radioactivity
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Cont….
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Using these and a few other techniques, the Genetic
Code was finally deciphered.
• Note: Genetic Code is Degenerate - a specific amino
acid may be specified by more than 1 codon
• Note: Genetic code is Unambiguous – each codon
specifies (or codes) for 1 and only 1 amino
acid
THE GENETIC CODE
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Characteristics of the genetic code (written as in
mRNA, 5’ to 3’):
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1. Code is triplet.
2. Code is non-overlapping
3. Code is non-Punctuating
4. Code is almost universal.
5. Code is unambiguous
6. Code is degenerate.
7. Code has start and stop signals.
8. Wobble occurs in the tRNA anti-codon
Wobble hypothesis
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ü Proposed by Francis Crick in 1966.
ü Occurs at 3’ end of codon /5’ end of anti- codon.
ü Result of arrangement of H-bonds of base pairs at the
3rd possition.
ü Degeneracy of the code is such that wobble always
results in translation of the same amino acid.
ü Complete set of codons can be read by fewer than 61
tRNAs.
Wobble hypothesis
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Wobble
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hypothesis
Pairing in 3rd
position of codons
is Less stringent.
In part, is due to
Inosine (I), which
can pair with U, C,
or A.
Wobble explains
degeneracy of code
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Genetic Code degeneracy
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4 different nucleotides (A, G, C, U)
20 amino acids
Wobble position is responsible for degeneracy
18 of 20 amino acids are coded by more than one codon. Met
and Trp are the only exceptions
Many amino acids are four-fold degenerate at the third
position
Phenylalani Leucin Serin Tyrosin S Cystein Tryptopha
ne e e e top e n
UU UUA UCU UA UAA UG UGG
U UU UCC U 24 UAG U
UUC G UCA UAC UGA UGC
UCG
Leucin Prolin Histidin Glutamin Arginin
e e e e e
CUU CCU CAU CAA CGU
CUC CCC CAC CAG CGC
CUA CCA CGA
CUG CCG CGG
Isoleucin Methioni Threonin Asparagin Lysin Serin Arginin
e ne e e e e e
AU AUG ACU AA AAA AGU AGG
U ACC U AAG AGC AGA
AUC ACA AAC
AUA ACG
Valin Alanin Aspartic Glutamic Glycin
e e Acid Acid e
GUU GCU GAU GAA GGU
GUC GCC GAC GAG GGC
GUA GCA GGA
GUG GCG GGG
Exceptions to the Code
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The genetic code is almost universal
Same codons are assigned to the same amino acids and to
the same START and STOP signals in the vast majority
of genes in animals, plants, and microorganisms
However, some exceptions have been found
ØExa: Mitochondrial genes
Mitochondrial genes
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Mitochondrial mRNA from animals or microorganisms
(but not from plants) is placed in a test tube ,
cytosolicprotein-synthesizing machinery (amino acids,
enzymes, tRNAs, ribosomes) fails to be translated into a
protein
The reason: these mitochondria use UGA to encode
tryptophan (Trp) rather than as a chain terminator
When translated by cytosolic machinery, synthesis
stops where Trp should have been inserted
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GENETIC CODE: Summary
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A. A codon (3 bases) specifies an amino acid
B. Sequential and non-overlapping
C. Degenerate (more than one codon/amino acid)
D. Some codons are start and stop signals
E. The code is nearly universal(see differences in
human mitochondrial code)
F. Sequences of bases in genes and amino acids in their
encoded proteins are colinear
G. Experiments with synthetic mRNAs established
codon assignments
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THANK YOU
