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A MILESTONE IN GENETICS: Cracking the Genetic Code

When scientists set out to decipher the genetic code in 1960, they knew that the four
letters of the genetic alphabetthe four nucleotides in RNAsomehow had to specify the 20
amino acids in the polypeptide gene products plus the initiation and termination of polypeptide
chains. A doublet codetwo nucleotides per amino acidwould not provide enough
information (42 = 16 codons). However, a triplet code seemed to provide too much information
(43 = 64 codons). Then, in 1961, Francis Crick and colleagues provided strong evidence for a
triplet code. But, which triplet codons specified which amino acids?
The first breakthrough came in 1961 when Marshall Nirenberg (1968 Nobel Prize
recipient) and J. Heinrich Matthaei demonstrated that synthetic RNA molecules could be used as
artificial mRNAs to direct in vitro protein synthesis.1 When ribosomes, aminoacyl-tRNAs, and
the soluble factors required for translation are purified free of natural mRNAs, these components
can be combined in vitro and stimulated to synthesize polypeptides by the addition of chemically
synthesized RNA molecules. If these synthetic mRNA molecules are of known nucleotide
content, the amino acid composition of the resulting polypeptides can be used to deduce which
codons specify which amino acids.
The first codon assignment (UUU for phenylalanine) was made when Nirenberg and
Matthaei demonstrated that polyuridylic acid [poly(U) = (U)n] directed the synthesis of
polyphenylalanine [(phenylalanine)m]. They used radioactively labeled phenylalanine (14Cphenylalanine) as a substrate in an in vitro translation system with poly(U), poly(C), and poly(A)
as artificial messenger RNAs. The


C-phenylalanine was incorporated into polyphenylalanine

only when poly(U) was used as the mRNA (Table 1). When each of 17 other labeled amino acids
was used in the poly(U)-stimulated translation system, no radioactivity was incorporated into

polypeptides. Given the triplet nature of the genetic code, Nirenberg and Matthaei concluded that
UUU must be a codon for phenylalanine. Shortly thereafter, poly(A) and poly(C) were shown to
encode polylysine and polyproline, respectively, allowing Nirenberg and colleagues to assign
codon AAA to lysine and CCC to proline. Poly(G) does not function as an mRNA in the in vitro
translation systems because base-pairing between the guanine residues results in complex threestranded structures.

Data are from Nirenberg and Matthaei, 1961. Proc. Natl. Acad. Sci. USA 47: 15881602.
Poly (I) is polyinosinic acid, which contains the purine hypoxanthine. Hypo-xanthine is like guanine in that it base-pairs with

Researchers in the laboratories of Nirenberg and Severo Ochoa (1959 Nobel Prize recipient)
extended the use of artificial mRNAs to synthetic copolymers with random sequences. For
example, Nirenberg and coworkers synthesized a random copolymer containing approximately
equal amounts of adenine and cytosine and used it as an artificial mRNA in their in vitro
translation system.2 A random AC copolymer composed of equal amounts of A and C will
contain 12.5 percent (1/ 2 1/ 2 1/ 2 = 1/ 8) of each of the eight possible codons: AAA, AAC,
ACA, CAA, CCA, CAC, ACC, and CCC. Nirenberg and colleagues observed that poly(AC)
directed the incorporation of six amino acidsasparagine, glutamine, histidine, lysine, proline,
and threonineinto polypeptides. Because they already knew that AAA and CCC were lysine

and proline codons, their results indicated that codons composed of two As plus one C and two
Cs plus one A specified asparagine, glutamine, histidine, and threonine. Approximately equal
amounts of asparagine, glutamine, histidine, and lysine were incorporated, but threonine and
proline were incorporated in about double the amount of each of the other four amino acids. This
result indicated that random AC copolymers might contain twice as many threonine and proline
codons as asparagine codons. Indeed, there are two threonine codons (ACC and ACA) and two
proline codons (CCC and CCA) in random AC copolymers, but only one codon for each of the
other four amino acids, for example, asparagine (AAC).
By varying the nucleotide composition of random copolymers, Ochoa and colleagues altered the
relative frequencies of the eight codons and looked for correlations with the frequencies of the
amino acids in the polypeptides synthesized in response to the copolymers.3 For example, they
synthesized random copolymers containing A and C in 5:1 and 1:5 ratios. When these
copolymers were used as mRNAs in vitro, the same six amino acids were incorporated as in
Nirenbergs experiments with a 1A:1C random copolymer, but their relative frequencies in the
polypeptide products were very different. With the 1A:5C copolymer, proline and lysine
represented 60.8 percent and 1.2 percent, respectively, of the amino acid residues incorporated,
in agreement with the earlier demonstration that CCC and AAA were proline and lysine codons.
The values were reversed when the 5A:1C copolymer was used; 51 percent and 3.8 percent of
the incorporated residues were lysine and proline, respectively. Ochoas results indicated that the
glutamine and asparagine codons contain 2 As and 1 C, whereas the histidine codon contains 1
A and 2 Cs, and so on.
A major breakthrough occurred when H. Ghobind Khorana (1968 Nobel Prize recipient) and
colleagues developed a procedure by which copolymers with known repeating di-, tri- and

tetranucleotide sequences could be synthesized.4 Because the codon sequences in these repeating
copolymers were fixed, their use as synthetic mRNAs yielded results that were more easily
interpreted than those obtained with random copolymers. For example, an RNA molecule with a
repeating UG dinucleotide sequence directed the synthesis of polypeptides containing alternating
cysteine and valine residues, as shown below.

Because the triplets UGU and GUG alternate in poly(UG)n, these two codons must specify
cysteine and valine, but the result does not tell us which codon specifies which amino acid. In
contrast, a repeating UUG trinucleotide polymer directed the synthesis of a mixture of
polyleucine, polycysteine, and polyvaline.

Because the initiation of translation occurs at random in these in vitro systems, some ribosomes
will translate these polymers as UUG, UUG, UUG, and so on, whereas others will translate them
as UGU, UGU, UGU. . . , and still others as GUU, GUU, GUU. . . . Thus, these three codons
must specify leucine, cysteine, and valine. Because the poly(UG)n product was a cysteine-valine
copolymer, one of the additional codons in poly(UUG)n, either UUG or GUU, must be a leucine
codon. By analyzing the amino acids incorporated in response to different repeating di-, tri-, and

tetranucleotide copolymers, Khorana and coworkers were able to assign many of the codons to
specific amino acids. For example, UGU specifies cysteine, UUG leucine, and GUU valine.
Additional information on the nature of the genetic code was obtained by assaying the binding of
aminoacyl-tRNAs to ribosomes activated with small RNA oligomers. When an in vitro
translation system is activated with poly(U), only one aminoacyl-tRNA, phenylalanyl-tRNAPhe,
binds to the ribosome. This specificity exists because the mRNA codon is part of the binding
site, and binding involves base-pairing between the codon and the anticodon of the tRNA. In
1964, Nirenberg and Philip Leder developed an assay for aminoacyl-tRNA binding to ribosomes
activated with trinucleotides, mini-mRNAs only three nucleotides long.5 Nirenberg and Leder
synthesized trinucleotides of known sequence and tested their ability to stimulate the binding of
specific aminoacyl-tRNAs to ribosomes. They assayed the ability of each trinucleotide to serve
as a mini-mRNA by using labeled amino acids to detect the formation of trinucleotideaminoacyl-tRNA-ribosome complexes (Figure 1). For example, the trinucleotides 5-UUU-3 and
5-UUC-3 both stimulated the binding of phenylalanyl-tRNAPhe to ribosomes, indicating that
UUU and UUC are both phenylalanine codons.
By combining the results of trinucleotide binding assays and in vitro translation
experiments performed with synthetic mRNAs, Nirenberg, Ochoa, Khorana, Leder, and others
were able to decipher the meaning of all 64 triplet codons (see Table 12.1). These codon
assignments are now firmly established, supported by definitive data from both in vitro and in
vivo studies.



The genetic code is largely the samethat is, each triplet codon has the same meaning

in all organismsviruses, bacteria, fungi, plants, and animals. Does this near-universality of the
code have any implications about evolution and the origin of species? If so, what? If not, how
can this near-universality of the code be explained?


If there are living organisms that store genetic information in nucleic acids and utilize

proteins for catalysis on other planets, do you think that they would use a genetic code identical
to ours? similar to ours? Why or why not?

Nirenberg, M. W., and J. H. Matthaei. 1961. The dependence of cell-free protein synthesis in E. coli upon naturally occurring or
synthetic polyribonucleotides. Proc. Natl. Acad. Sci. U.S.A. 47: 15881602.
Nirenberg, M. W., O. W. Jones, P. Leder, B. F. C. Clark, W. S. Sly, and S. Pestka. 1963. On the coding of genetic information.
Cold Spring Harbor Symp. Quant. Biol. 28: 549557.
Speyer, J. F., P. Lengyel, C. Basilio, and S. Ochoa. 1962. Synthetic polynucleotides and the amino acid code, IV. Proc. Natl.
Acad. Sci. U.S.A. 48: 441448.
Khorana, H. G., H. Bchi, H. Ghosh, N. Gupta, T. M. Jacob, H. Kssel, R. Morgan, S. A. Narang, E. Ohtsuka, and R. D. Wells.
1966. Polynucleotide synthesis and the genetic code. Cold Spring Harbor Symp. Quant. Biol. 31: 3949.
Nirenberg, M., and P. Leder. 1964. RNA codewords and protein synthesis: The effect of trinucleotides upon the binding of
sRNA to ribosomes. Science 145: 13991407.