Genetic Code

2
Learning Objectives
Genetic Code: Genetic Code is a Triplet Code
Deciphering of Genetic Code
Deciphering of Genetic Code
Essential Features of Genetic Code
Genetic Code is Universal
Cracking of Genetic Code

The linear arrangement of nitrogenous bases in DNA is said to determine the

sequence of amino acids in a protein molecule. There are only four nitrogenous
bases in a DNA molecule, namely adenine (A), guanine (G), cytosine (C) and
thymine (T). It means the precise sequence of these four nitrogenous bases on
the DNA strand somehow direct the proper amino acids to their proper places
through the intervention of mRNA.

12.1 Genefic Code

Several theories were proposed to explain the mechanism by which a particular

sequence of nitrogenous bases in DNA, by transcribing complementary bases in
mRNA, determines the position of specific amino acid in the protein molecule.
The theory which is widely accepted till now was proposed by F. H. C. Crick.
The theory holds the existence of a genetic code and its smallest unit which codes
for one amino acid is known as codon.
A codon (code word) is the nucleotide or nucleotide sequence in mRNA which
codes for a particular amino acid, whereas the genetic code is the sequence of
nitrogenous bases in mRNA molecule which encloses information for the synthesis
of protein molecules.

Genetic Code is a Triplet Code

The main problem of genetic code was to determine the exact number of nucleotides
in a codon which codes for one amino acid. Since there are only four nitrogenous
bases in mRNA for 20 amin0 acids, combination of only one or two nitrogenous
bases can not provide sufficient code words for 20 amino acids. A singlet code

273
 274 Principles of Molecular Biology

AC AG AU
FIGURE 12.1 A AA
CG
CA CU
CC
Codons of singlet Codons GA GC GU
GG
and doublet codes of
UU UA UG UC
nitrogenous base
4 x 4 = 16 codons
Doublet Code:
Singlet Code: 4 x 1 = 4 codons

four codons A, C, G and U. The

consisting of only one nucleotide provides just These
Ala Alanine acids. Similarly a
combination nitrogeno
of two
enous
are insufficient to code for 20 amino insufficient for 20
Arg Arginine x 4 16 codons still amino
bases (doublet code) provides 4 =

Asn Asparagine
acids.
Asp Aspartic acid of three letter code, i.e. each codan
Cys Cysteine
Gamow (1954) pointed out the possibility
consisting of three nitrogenous bases.
GIn Glutamine This will give 4 x 4x 4 64 code words or codons, which are more than enough
Glu Glutamic acid to code for twenty amino acids. The Table 12.1 provides the list of 64 triplet codone
Gly Glycine
for amino acids.
His Histidine It is evident from the table that several of the triplets have the same letters but
lle Isoleucine in different sequences and these code for different amin0 acids. It means that the
Leu= Leucine sequence of letters in the triplets is most important in determining what amino aci
Lys Lysine is to be coded.
Met Methionine Although, information are coded in the form of nitrogenous base sequences in
Phe Phenylalanine
DNA molecule, it is customary to represent the code letters of mRNA because the
Pro Proline message from DNA is carried into the cytoplasm by mRNA and the code on mRNA
Ser Serine chain.
is translated into the sequence of amino acids in the polypeptide
Thr Threonine
Trp Tryptophan
Tyr Tyrosine Deciphering of Genetic Code
Val = Valine
By the process of in vitro synthesis, scientists have found out codons for all the
twenty amino acids. These codons have been listed in Table 12.2.

TABLE 12.1 Triplet codons of mRNA for amino acids

C A G
U
UUUPhe UCU UAU
UAC Tyr
UGU
UGCCys
UUC UCC C
UCA Ser A
uUA
IUG
Leu UCG UAA Ochre Terminator)
UAG Amber (Terminator)
UGA
UGG Trp
Terminator G

CUU CCU
CAU His
CAC His
CGU
CUC
CUA Leu CCCPro
CCA
CGC
CGA Arg
CUG CCG CAA Gin
CAG Gin CGG G

A AUU ACU

AUA
AUC Ile ACC
ACA
Thr AAUAsn
AAC AGU Ser
AGC Ser
ACG AAA LyS
AAG Lys AGA Arg
AGG Arg
G
AUG Met Initiation codon

G GUU
GUC
GCU
GCC
GAU ASP
GACAspp
GGU
Val Ala GGC
GUA GCA GGA Gly A
GUG GCG GAA Glu
GAG GGG G
 Genetic Code 275
Chapter 12

TABLE 12.2: Triplet codons for amino

Amino acid
Abbreviation acids mRNA codon
1. Alanine
DNA Code
ala
CGA, CGG, CGT, cGC GCU, GCC, GCA, GCG
2. Arginine
arg GCA, GCT, GCC, TCT, GCG, TCC AGG
3. Asparagine
CGU, CGA, CGG, cGC, AGA,
asn
TTA, TTG3 AAU, AAC
4. Aspartic acid
asp CTA, CTG GAU, GAC
5. Cysteine
cys ACA, ACG UGU, UGC
6. Glutamine
gln GTT, GTC CAA, CAG
7. Glutamic acid
glu CTT, CTC GAA, GAG
8. Glycine gly CCA, CCG, CCT, CCc GGU, GGC, GGA, GGG
Histidine his GTA, GTG CAU, CAC
10. Isoleucine ile TAT, TAA, TAG AUA, AUU, AUC
11. Leucine leu AAT, AAC, GAA, GAG, GAT, GAC UUA, UUG, CUU, CUC, CUA, CUG
12. Lysine lys TTT, TTC AAA, AAG
13. Methionine met TAC AUG
14 Phenylalanine phe AAA, AAG UUU, UUC
15. Proline CCU, CCC, cCA, CcG
pro GGA, GGG, GGT, GGC
16. Serine ser AGA, AGG, AGT, AGC, TCA, TCG UCU, UCC, UCA, UCG, AGU, AGC
17. Threonine thr ACU, ACC, ACA, ACG
TGA, TGG, TGT, TGC
18. Tryptophan trp ACC UGG
19. Tyrosine tyr ATA, ATG UAU, UAC
20. Valine val CAA, CAG, CAT, CAC GUU, GUC, GUA, GUG
21. Terminating triplets ATT, ATC, ACT UAA, UAG, UGA

12.2 Nature and Characteristics of Genetic Code

The characteristics of genetic code are as follows:

1. Genetic Code is a Triplet Code

A codon of the present day genetic code that specifies one amino acid in a polypeptide
chain comprises of a sequence of three nitrogenous bases on mRNA in a specific
sequence.

2. Genetic Code is Commaless

Genetic code on mRNA is read continuously without internal punctuation along the
the adjacent codons and each
reading frame. It means there is no comma between
codon is immediately followed by the next codon with no intervening spaces for
comma and no skipping of any nucleotide in the message.

3. Genetic Code is Non-overlapping

The codons in mRNA are read in successive groups of three nucleotides in 5 ' 3 '
direction. A message of AAGAAGAAG.. n mRNA will code for lysine-lysine-
for lysine. Since, there is no comma, theoretically
lysine ....because AAG codes
the reading of message can begin as AAG or AGA or as GAA depending upon
shown below:
where the reading begins as
276 Principles of Molecular Biology

described as overlapping sequence rea

message is
Overlapping code This type of reading of that translation of genetic code
available to show
AUA CG AGU But evidences ie.. oe
Trame. are
there is no overlapping of codons,
Tyr mRNA begins at the correct point and genetic
UAC code is nonoverlapping.
Tyr
ACG
Thr
4. Genetic Code is Unambiguous
aid to be nonambigitos
GA medium (in vivo) is uous
The genetic code inside the cell doubt th amino acid. No
Arg codon always codes for
same the
same aminoparticular
because a
acid may be coded by more than one codons (degeneracy), but ona
codon never codes for two different amino acids.
In certain rare cases the genetic code is found to be ambiguous (same codon
different conditions). For example, in
coding for different amino acids under
streptomycin sensitive strain of E. coli, UUU codon, normally codes for phenylalanine
but may also code for isoleucine, leucine or serine when ribosomes are treated with
streptomycin. Another example of ambiguity is seen when GUG, sometimes, functions
as a start codon in protein synthesis. As a start codon GUG specifies amino acid
methionine, whereas GUG within the coding sequence codes for valine. This is
called site specific variation in codon translation and is also described as context
effect in codon translation. This ambiguity is enhanced at high Mg-ion concentration.
low temperature and in the presence of ethyl alcohol.

5. Genetic Code is almost Universal with Few Exceptions

The same genetic code is said to be present in all kinds of living organisms including
viruses, bacteria, unicellular and multicellular organisms. Thus, for example, lysine
is coded by AAA or AAG in the mRNA of all organisms, arginine by CGU, CGC,
CGA and so on. Hence, scientists can isolate mRNA from one organism, translate
it by using synthetic machinery isolated from another organism and obtain proteins
as if it has been synthesised in the original organism. However, code is not absolutely
universal. There are a few differences in the genetic code in mitochondria, chloroplasts
and some organisms. Protein synthesis in plant mitochondria uses the universal
nuclear genetic code. But, mitochondria of other organisms have differences in their
codons shown as under:
1. AUA and AUG both code for methionine, instead only AUG is initiation
codon and codes for methionine. In nuclear code AUA
specifies isoleucine.
2. UGA in the nuclear code acts as a codon
stop but in mitochondria it specities
tryptophan.
3. AGA and AGG are stop codons in mitochondria but
in the nucleus.
act as arginine codons

4. UAA and UAG do not act as

stop codons in mitochondria of some ciliated
Protozoa. They code for glutamine.

6. Genetic Code is Degenerate

Since there are 4= 64 possible codons for
only 20 amino acids that contribute
proteins, it was concluded that in
a most cases
single amino acid is coded by tw
to several different codons. This
multiple system of coding
system or degenerate genetic code. Except tor represents degene
have only one codon each, all methionine and tryptophan, W
other amino acids have two or more codons. e
degeneracy of coding system prOvides protection to
mutations, stabilises phenotypes by lessening the organisms against many harn
effect of random mutations a
 Chapter 12 Genetic Code 277

TABLE 12.3: Differences between human and yeast mitochondrial genetic codE
S.No.
Codon Nuclear Code
Amino Acid
Mitochondrial code in human Mitochondrial code in yeast
UGA
Termination Tryptophan Tryptophan
AUA
Isoleucine Methionine Isoleucine
CUN
Leucine Leucine Threonine
AGG, AGA
4. Arginine Termination Arginine
5. CGN
Arginine Arginine Termination

ail 1our
in codon for amino acid
Note: "N
leucine.
represenis nitrogenous bases A,G,C.U. Any one of these nitrogenous bases can be present

minimises the consequences of base pairing errors. Codons that specify the same
amino acid are called synonymous. Most synonyms differ only in their third base
ofthe codons. For example, GUU, GUC, GUA and GUG all code for amino acid
valine. It means the major degeneracy occurs at the third position at the 3' end of
the triplet codon. When first two bases are specified, the same amino acid may be
coded for whether the thi base is U, C, A or G. This base is described as *Wobbly
base'.
For example, note the gentic codes for the following amino acids:
.The codons that
Serine: UCG, UCC, UCA, UCG and AGU, AGC specify the same
amino acids are said
(b) Argine: CGU, CGC, CGA, CGG and AGA, AGG
to be synonymous.
(c) Leucine: CUU, CUC, CUA, CUG and UUA, UUG
Different tRNAs that
(d) Valine: GUU, GUC, GUA, GUG accept the same

The following table represents the occurrence of multiple codons for different amino acid but have
different anticodons
amino acids and clearly illustrates the degeneracy of genetic code: are called
isoaccepting tRNAs.
No. of codons for Total no. of
S.No. Amino acids each amino acid codonsS
1. Arginine, Leucine, Serine 6 each 18
Alanine, Glycine, Prolne, 4 each 20
Threonine, Valine
Isoleucine, Stop codons 3 each
3.
2 each 18
4. Asparagine, Aspartic acid, Cysteine,
Glutamic acid, Glutamine, Histidine
Lysine, Phenylalanine and Tyrosine
1 each 02
Methionine and Tryptophan
64
Total

Extension: Genetic Code

codons or termination codons (UGA, UAA, UAG) and
4 64. Out of these, three are stop
Genetic code contains 64 codons Crick's wobble hypothesis, only 32
60 codons code Tor
20
amino acids. According to
one initiation codon (AUG). The remaining nature of first base of anticodon.
sufficient to transport
20 amino acids, no
the
60. Because of wobbling
ypes of tRNA could be for the same amino acid.
different codons coding
R N A can recognise several
278 Principles of Molecular Biology

Reasons for Degeneracy: Wobble Hypothesis

(Wobble Occurs in the Anticodon)
Wobble or Fluctuating Base: Crick (1966) proposed Wobble hypothesis. According
on tRNA Is usually an
to this hypothesis, the base in first position of anticodon
These abnormal bases are
abnormal base, like inosine, pseudouridine, tyrosine, etc.
able to pair with more than one type of nitrogenous
bases in the third poSition of
C or U. This base
with A,
the codon on mRNA. For example, inosine (I) can pair
is called a Wobble base or fluctuating base.
to
The last two nucleotides of an anticodon on tRNA are strictly complementary
first two nucleotides of codon in mRNA as per classical rule of pairing. But the
less stringent
pairing of third base on mRNA with first base of anticodon of tRNA is
and may occur in a nonclassical fashion, so that in some cases a single tRNA may
base pair with more than one codon. This is called Pairing Wobble or Wobble
Base Pairing.

Wobbling between anticodons and codons allows some tRNA molecules to read more than one codon.

The codon/anticodon base pairing rules are known as Wobble rules.

TABLE 12.4: Anticodon codon base pairing rules and wobble pairing
obble base pairing with nucleotide
Nucleotide at first position at the 5 Normal pairing with nucleotide at 3 in mRNA
end of tRNA anticodon position of codon at 3 end of mRNA
G C Uor C
G G
A U
U A or G
I (Inosine A, U or C

FIGURE 12.2 Leucine codons

Leu Leu
Two examples of wobble
Identical 3
base pairing: 5 lenucine
A. Two different leucine
codons CUC and
CUU can be read by
tRNAs
JO
the same anticodon
of leucine-tRNA; GAG Normal GAG WNobble
B. Three different pairing pairing
mRNA C UC
glycine codons GGU 5' L3'
GGC and GGA can
be read by the same Glycine codons
anticodon of glycine. Gly Gly Gly
tRNA molecule 3 ldentical
having abnormal or 9lycine
wobble base (1).
ooc- tRNAs

CCI
n o sInosine
ine C

GGC GA

B
 D

E
D

C i

9
280 Principles of Molecular Biology

Marshall Warren Nirenberg (April 10, 1927-Jan. 15, 2010) H.G. Khorana (Jan. 9, 1922-Nov.
9, 2011)
Nirenberg. an American biochemist Har Gobind Khorana was born in 1922 in
and geneticist successfully synthesised Raipur, Chattisgarh. He made remarkable
protein in cell-tree protein synthesising contribution in synthesis
artificial of nucleic
obtained from E. coli. acids. He discovered how to synthesise
system
By adding
new mRNA (poly U), Nirenberg was triplet RNA molecules of known sequence
able to synthesise phenyl alanine the genetic code.
thereby assigning
polypeptide chain. This was described He also synthesised artificial genes (long
as cracking of genetie code. It showed DNA molecules). Khorana shared in
1968
that UUU coded for phenylalanine. For Nobel Prize with Marshall W. Nirenberg
this discovery he shared 1968 Nobel and Robert W. Holley.
Prize along with H.G.Khorana.

studies of mutants. These head protein molecules. These

produce incomplete
Ta
mutants can be shown to map in linear sequence by the recombination technique.
This suggests that the code is collinear.

12.3 Discovery of Genetic Code or Cracking of Genetic Code

The existence of a triplet code was simply an assumption till Marshall Nirenberg
(Nobel Prize winner) and Heinrich Matthaei in 1961 proved its existence by

experiments. They wereable to synthesise artificial mRNA which contained molecules

of only one base uracil. It was named polyuridylic (poly U) molecule. The synthetic

poly U mRNA was placed cell-free system containing protein-synthesising

in a
Escherichia coli and
system, enzyme polynucleotide phosphorylase extracted from
the twenty amino acids together with necessary ATP. After some time a small
protein-like molecule was produced which was formed by the linking of phenylalanine
(a homopolymer).

Nirenberg and Matthaei Initially used Homopolymers

In their initial experiments, Nirenberg and Matthaei used RNA homopolymers of
each type of ribonucleotides, like homopolymers of U(UUUUUU..), C (CCCCCc.)
and of G (GGGGG...). They found that poly U produced polyphenylalanine
polypeptide, poly A resulted in the synthesis of polylysine and poly C produced
polyproline. On the basis of above information codons UUU, AAA and CCC were
assigned for amino acids phenylalanine, lysine and proline.

Use of Mixed Copolymers

After establishing the codons of homopolymers, Nirenberg, Matthaei and Ochoa
used RNA heteropolymers. Using all the four ribonucleotides to construct synthetic
mRNA, they established the nature of codons formed by two or more nitrogenous
bases, such as ACA, CCA, CAA, etc. These were described as copolymers. Finally
by the end of 1968, composition of all the 64 triplet codons corresponding to all
20 amin0 acids were established.

Triplet Binding Assay

In 1964, Nirenberg and Philip Leder developed the triplet binding assay. For
this, triplets of known sequences were synthesised in the laboratory to serve as
templates. The radioactively charged tRNA, the RNA triplets and ribosomes were
 Chapter 12 Genetic Code 281

TABLE 12.5: List of synthetic co-polymers of RNA, codons derived from them ana
amino acid incorporated to form polypeptide
Copolymers Codons formed Coded for amino acid in polypeptide chain

UG
UGU Cysteine
GUG Valine

AC ACA Threonine
CAC Histidine

UUC UUC Phenylalanine

UCU Serine
C JU Leucine

AUC AUC Isoleucine

UCA Serine
CAU Histidine

UAUC UAU, UAC Tyrosine

CUA, UUA, CUU Leucine
UCA, UCU Serine
AUC, AUU Isoleucine

GAUA GAU Aspartic acid

AGA None
UGA None (Termination)
UAG None (Termination)
AUA Isoleucine
UAA None (Termination)

incubated on a nitrocellulose filter and radioactive tRNA associated with RNA triplet,
and ribosomes were isolated from the nitrocellulose filter and were identified. This
revealed the specific codon and anticodon association.

Contribution of Khorana
Indian biochemist, Dr. H.G. Khorana, devised an ingenious technique for artificially
synthesisingmRNA with repeated sequences of known nucleotides. For this valuable
contribution he was awarded Nobel Prize in 1968.
Khorana and his coworkers prepared chains of
By using synthetic DNA,
with known repeating sequences of two, three and four nucleotides
polyribonucleotides
as follows:
Poly CUC UCU CUC UCU

Poly CUA CUA CUA CUA

two codons arranged alternately in the polynucleotide
In first case CUC and UCU are
chain. This dictates the formation of polypeptide chain having two amino acids
The second case is an example of
(leucine and serine) arranged alternately.chain is formed of
homopolymer chain. The polynucleotide repeated linkage of
codon CUA. This dictates the formation of a polypeptide chain consisting of only

one amino acid leucine.

282 Principles of Molecular Biology

TEXT QUESTIONS
What do you mean by "Genetic code? Discuss in brief the special Teatures of genetie

code.
code.
Describe the contribution of Dr. H.G. Khorana in understanding genetic

3. What is cracking of the genetic code?

4. Outline the essential features of triplet code.
5. Write short notes on:
H.G. Khorana
(a) Contribution of Kornberg (b) Contribution of
(c) Ambiguity (d) Collinearity.
6. The triplet genetic code is degenerate. How could you justify the statement?

7. It is found that both

(a) UUU and VVC code for phenylalanine
(b) But when ethyl alcohol is used in high concentration, the incorporation of leucine
and isoleucine is sharply increased, while the incorporation of phenylalanine is
decreased for the same codons.
Which of these situations (a) or (b) represents ambiguity and which degeneracy'
8. What is the role of nonsense codons in protein synthesis?
9. Who coined the term 'genetic code'?
10. What is codon?
11. What is anti-codon?
12. What is genetic code?
13. Name three nonsense codons.
14. How many bases code for one amino acid?
15. Name the amino acids which have one codon each.
16. Genetic code is non-overlapping and degenerate, why?
17. Name two codons that code for more than one amino acid.
18. Which base triplets code for the amino acid phenylalanine?
19. Are there any base triplets that code for amino acids and also for start signals?
20. What is the role of nonsense codons in protein synthesis?
21. Which RNA molecule bears codons and which RNA molecules has anti-codon?
22. What anti-codons will be required to recognise the following codons
(a) AAU (b) CGA
23. Who cracked the genetic code?
24. Describe biological significance of degeneracy of genetic code.
25. What is the use of synthetic copolymers?
26. Why is the concept of "universality of genetic code' threatened?