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Genetic Code

It refers to the relation between the four-letter nucleotide

sequence information in mRNA to the amino acid
sequence information in proteins.

The cracking of the genetic code is a history now.

Several theoretical considerations and elegant

experimental results supported that a group of three
nucleotides, called a codon, specifies an amino acid.

Codon is a group nts specifying one aa

How many codons are possible for 20 naturally

occurring acids ?

Researchers were trying to decipher genetic code

before even mRNA was isolated .
Theoretically the number of nucleotides in a given codon could be 1,
2, 3 or 4
Codons with one nt. Then 4 nt specify 4 codons 41 = 4
If each codon specifies one aa, then 4 codons specify 4 out of 20 amino
acids (aa).

Codons with two nts specifying one aa, then 42 = 16 codons specify 20

Codons with three nts specifying one aa 43 = 64 codons specify 20 aa

Codons with Four nts specifying one aa, then 44 = 256 codons specify
20 aa

The single and double letter codons cannot represent all of the twenty
amino acids.

Codons containing 4 nts was also not supported experimentally and

theoretically as it uses maximally of the 4 letters to specify 20 aa
compared to the triplet code.
Exptl evidence backing theoretical considerations suggest that a
codon comprising three nts may be specifying an aa because the
very early mutagenesis experiments involving bacteriophage T4
had shown that the virus was unable to tolerate genetic
changes that probably have altered one or two bases. However,
the phage was able to tolerate an alteration (deletion or
insertion) of three bases. Based on such genetic experiments, it
is suggested that the genetic code is a triplet code.

In a triplet code, a change in one or two bases (insertion or

deletion) alters the reading frame of the mRNA and all the
subsequent amino acids that are incorporated into the protein to
the right side of deletion (-) or insertion (+) (that is on the –COOH
side of protein). However insertion of three nucleotides into an
mRNA sequence would change the coding ability of the message
only of those triplets at, and between the insertion of bases but
not the amino acid sequence downstream of the three inserted
bases .
Example: An mRNA with the following sequence with 7 codons each
having 3 nt
Polypepetide of 7 aminoacids – may be all of them are similar because
the codons contain the same nts


If one nt in one of the codons ( say B in third codon) is deleted

2 nt deletion :

3 nt deletion

The changes would be more drastic if one or two nt are deleted compared to
deletion of 3 nts. Similarly, insertions of nts. Insertion of 3 nts may change one
aa and that is perhaps more tolerable than several aa
Cell free translational systems translate given messenger RNA to the corresponding protein
in test tube reactions in the presence of a cell extract that is devoid of its DNA but contains
the necessary translational machinery like ribosomes, tRNA, amino acyl synthetases that
couple amino acids to the the respective tRNAs .

The extracts are supplemented with salts like K+ and Mg2+ , ATP, GTP etc., necessary for
protein synthesis.

To determine the amount of protein synthesis, one of the 20 amino acids will be labelled
which can be incorporated into a protein.

At the end of protein synthesis, few microliters of the reaction mixture will be spotted on a
filter paper, and is then dipped in 10% ice cold tricholoroacetic acid that precipitates protein
in the reaction mixture which is stuck to the filter paper.

Filter paper is washed with 5% hot tricholoroacetic acid (TCA) to remove any non specific
radioactivity that is bound to the filter paper. Afterwards the filters are washed with
hypochloric acid to remove any color that comes from the extract and is bound to the filter
paper (ex: in the case of red blood cell extracts)

To remove the water and dry the filters, the latter are washed with choloroform and ethanol
three-four times and are air dreied. The filters are then placed in few ml of scintilation fluid
and are read in a scintillation counter to determine the radioactivity bound to them.
Cell Free Translational Systems are used to decipher the genetic code
Cells are lysed, DNA/ nucleus is removed as it contains transcriptional
apparatus by treating the extracts with DNAses. Endogenous mRNAS can
also be removed by treating with micrococal nuclease and Ca2+ in order to
test the effect of exogenous mRNA. Prior to adding mRNA the micrococal
nuclease can be inactivated by EGTA that removes Ca2+

the cell-free protein synthesizing systems contain all the machinery viz.,
the ribosomes, transfer RNAs, aminoacyl synthetases that catalyze the
joining of amino acids to tRNAs and other soluble factors (initiation,
elongation and termination factors) required for the protein synthesis.

Additionally the extracts are supplemented with ATP, GTP, and energy
generating enzymes along with salts like K+ and Mg2+. Such cell-free
translational systems derived from bacterial extracts eventually provided a
means to determine the amino acid sequence of proteins coded by artificial
and natural templates.

The protein synthesis of the extracts carried out by endogenous mRNAs,

or by exogenously supplied mRNAs can be monitored by the incorporation
of radioactively labeled amino acid into the acid precipitable protein.
Uses/ applications of cell free translational systems.
a) as a source for the preparation of reconstituted lysates to identify
defective components of translational machinery,
b) for the purification of protein factors and enzymes, to study their
c) evaluate the mechanisms of actions of different antiobiotics on various
steps in protein synthesis
d) to determine the mechanics and regulation of cytosolic and secretory
protein synthesis.

Since many proteins are processed in physiological conditions and is

difficult to know the sequence if any in prepro-proteins (unprocessed
proteins), translation of such mRNAs in vitro systems devoid of the
peptidases or proteases has provided a means to determine the sequence
of full length proteins.

In addition, these translational systems are found useful to evaluate the

effects of toxins, and a host of other novel compounds for their ability to
affect protein synthesis at specific steps.

To identify a membrane bound protein from a non membrane bound

protein by following its transport after its synthesis. This can be achieved
by supplementing liposomes to the cell free extracts and following the
transport of a protein to the liposomes
Artificial/synthetic mRNA templates were used as natural mRNAs
were not yet purified or they know the sequence of them to relate the nt
sequence to aa sequence.

The discovery of polynucleotide phosphorylase enzyme by Ochao

and Manago.

This enzyme is found to catalyze in fact the degradation of RNAs to the

corresponding ribonucleotides and inorganic phosphate in vivo. But,
interestingly, the purified enzyme has been observed to catalyze
the formation of ribonucleoside triphosphates (rNTPs) in the
presence of high concentration of ribonucleoside diphosphates in
The incorporation of a ribonucleotide (A, U, G or C) is however
dependent on the relative amounts of the nucleotides used in the
reaction mixture.

For example, the chances of the incorporation of nucleotide A

(adenosine) only once in the template molecule in a reaction mixture
consisting of A and C in the ratio of 1: 4 are 1/5 (~20%) whereas the
chances for the incorporation of C are 4/5, (~ 80%) the highest.

In other words, a template molecule with AAA will be synthesized least

(1/5x1/5x1/5= 1/125= 0.8%) and CCC (4/5x4/5x4/5= 64/125=51.2%) will
be the most abundant .

With random incorporation of bases, the probability of any of the

codons with 2 Cs and one A ( CCA, ACC, CAC ) would be 4/5 x4/5 x1/5
= 16/125 = .128 or 12.8%

The probability of any codon with two As and one C (AAC, ACA, CAA)
would be 1/5 x 1/5 x 4/5 = 4/125 = 0.032 or about 3%
Amino acid analyses of polypeptides produced by
synthetic templates
Synthetic Homopolymers and their translation yielded the
following :

AAAAAAAAAA--- Polylysine
CCCCCCCCC- polyproline
GGGGGGGGG – polyglycine but synthesis is weak as
polyglycine cannot properly fold

Translation of synthetic alternating nts:

ACACACACAAC--- yields a polypeptide with alternating
threonine and histidine aa

It is not clear whether ACA or CAC has yielded histidine or

Templates with Repeating trinucleotides


Such sequences produced three types of polypepetides:

1.All with Aspargine,
2. All with threonine,
3. All with glutamine
This is possible if the start site in the mRNA in each of these cases is


AACAACAACAACAAC (ACA) (if first A is skipped)

AACAACAACAACAAC (CAA) (if first two nts are skipped)

The common amino acid that is incorporated when the mRNAs containing
AC repeats and AAC repeats is threonine.

Based on such results it is suggested that ACA codon may be responsible

for the incorporation of the amino acid threonine.
The reading frame in natural mRNAs is set by start codon which is
usually AUG and the code is generally nonoverlapping or in other words
each nucleotide in an mRNA belongs to a single reading frame.

Codon, charged tRNA and ribosome complexes can be captured on a

Researchers who were in the race to crack the genetic code had yet another
interesting observation wherein they found that a small triplet codon (like
AUG (Met) , AAA (lys)or CUG (Leu)) etc., can attract a complementary
anticodon of a tRNA carrying the amino acid.

The codon and anticodon together can bind to ribosomes.

Such codon-charged tRNA-ribosome complexes can be captured on a filter

Triplet codon + tRNA with its amino acid + ribosomes can form a complex
and can be retained on a filter paper.
This technique is found to be relatively more informative in deciphering the
genetic code because it is easy to prepare and handle small triplet
nucleotide sequences than long RNA templates.

The synthetic triplet codons were incubated with a mixture of tRNAs each
carrying a radioactive amino acid.

Small molecules like the tRNAs carrying amino acids whose anticodons are
not complementary to the codons cannot bind to the codon in the reaction
and will pass through the filter.

This technique really facilitated to identify the recognition of 61 triplet codons

that are now known to recognize the twenty naturally occurring amino acids

Out of the total 64 codons, 61 codons specify 20 amino acids, and

three codons UAA, UAG and UGA specify stop codons.

Since 61 codons specify the 20 naturally occurring amino acids, it

suggests that there are many amino acids that are specified by more
than one codon. Hence it is suggested that the genetic code is
degenerate. ((that means an amino acid is specified by more than one
No of tRNAs do not correspond to sense codons and wobble:

Based on the number of sense codons, it is suggested that there should be

61 tRNAs recognizing the 61 sense codons.

Purification and characterization of tRNAs revealed that many purified

tRNAs a) do not correspond to no of sense codons, b) recognize more than
one amino acid, and c) tRNAs contain some modified bases like inosine
(derived from adenine) in the anticodon that is different from the regular four
bases present in RNAs.

Transfer RNAs with different anticodons that accept the same amino acid
are called isosccepting tRNAs. Codons specifying same aa are said to be
synonymus codons

These observations suggest that the first base in the 5’-anticodon of tRNA
does not follow strict Watson-Crick base-pair rule position while pairing with
the 3’ end of codon.

Wobble: This imperfect or nonstandard base pairing called wobble occurs

between the third position of the codon in mRNA and the first position of the
anticodon in the tRNA
Imperfect Base Pairing (Wobble): Nonstandard
base pairing between the 1st position of the
anticodon (5’side) with the third position in the
codon (3’ side).

Bases in an mRNA 3’ CC A ACA 5’

C A G U ( bases in the third position of codon ( mRNA) )

G U C A ( Bases in the first position of anticodon (tRNA)

C A G U I (first /wobble position of tRNA anticodon

G U C A C ( base in 3 rd position of mRNA codon)
C A G U I ( first or wobble position of
C A G U ( bases in the third position of codon ( mRNA) ) anticodon recognizes)
G U C A ( Bases in the first position of anticodon ) G U C A C ( base in 3 rd position of codon)
Analyses of AA codons
Analyses of the Codons in the Genetic Code:

Total 64 Codons
Stop Codons Three, UAA, UAG and UGA
Sense Codons Sixty One,

Three amino acids: Leu, Ser and Arg Six Codons each, Total = 18 Codons

Five aa: Gly, Pro, Ala, Val, Thr Four codons each, Total= 20 Codons

One aa: Ile Three Codons, Total = 3 Codons

Nine aa: Phe, Tyr, Cys, His, Glu, Gln, Asn, Two Codons each, Total = 18 Codons

Asp, Lys

Two amino acids, Met, Trp One codon each , Total = 2 Codons

Total No. Sense Codons 61 Codons

Variations from the Universal genetic Code in different species

Mitochondrial DNA in
Human and yeast UGA codes Trp instead as a stop codon
Human AUA codes Met instead Ile
Human AGA and AGC code Stop codon instead Arg

Nuclear DNA in
Tetrahymena UAA codes Gln instead as a stop codon
Paramecium UAG codes Gln instead as a stop codon

Changes in the nucleotide sequences alter the genetic code.

Point mutations arise because of changes in a single nucleotide which are
different from other kinds of drastic changes that occur in DNA due to extensive
insertions and deletions

Missense mutation / Substitution mutation

A change in one codon (trinucleotide sequence) that specifies a particular amino

acid to another codon that specifies a different amino acid can occur by a single
base change. The best example is sickle cell anemia, a human genetic disease.
Here the glutamic acid at position six in the human β-globin is replaced by
valine. The change is the result of a base change at the second position of the
codon GAA that codes for glutamic acid to GUA that codes for valine.

Nonsense mutation
A change in the base of a codon specifying an amino acid to another base that
results in a stop codon. If it happens in the middle of a genetic message, it results
in premature termination and incomplete polypeptide synthesis.
Silent mutation
Often a change in the base that is present in the third position of a codon of an mRNA
to another base can result in a synonym that does not alter the amino acid sequence
of the encoded protein. Such changes are called silent mutations.

Neutral Mutation
It is a kind of missense mutation where the change in a base leads to a change in the
incorporation of a different amino acid that is chemically similar to the original one,
and does not influence the protein function.

Frame shift mutations

Insertion or deletion of one or more base pairs in the gene sequence affects the
reading frame of mRNA. As a consequence the amino acid sequence of the protein
from the point of mutation to the C-terminus will be modified. For example 5’…. ABC
ABC ABC ABC ABC ABC ……3’ sequence in mRNA is modified by the insertion of a D for
example can give a rise a message to ABC ABC DAB CAB CAB CAB C. Even deletion of a
base also alters the coding capacity not only at the site of modification but also the rest
of the message from the site of modification. However in the above sequence if
insertion or deletion of three bases (like D, E and F) instead of one or two bases occurs
then the coding specificity of the message is altered at and between the insertions of
these bases but the downstream sequences are not altered as shown below.
5’…. ABC ABC ABC ABC ABC ABC ……3’ is changed to 5’…. ABC DAB CAE BCA FBC ABC
ABC ……3’.
Suppressor Mutations

A suppressor mutation as the name indicates, suppresses the influence of

another mutation.

Thus the organism that contains a suppressor mutation is a double mutant

and it has two mutations: one the original mutation and the second one
being the suppressor mutation that will produce a phenotype which is
similar to the wild type.

However this is not called reverse mutation. In reverse mutation, whatever

change occurred in the original nucleotide is restored. A suppressor
mutation occurs away from the original mutation site. The suppressor
mutation can occur within the same gene that contains already a mutation
(intragenic suppressor) or in another gene (intergenic suppressor). Both
kinds of suppressor mutations block the effects of an earlier mutation.