Cell-free Translation Systems
453
groups. The addition of viral genomic RNA
resulted in a large increase of amino acid
incorporation into polypeptide, concluding
that RNA serves as a template for protein.
1.2
Determination of the Genetic Code
One of the most remarkable contributions
of the cell-free translation system in molec-
ular biology was the determination of the
genetic code in the 1960s. Matthaei and
Nierenberg demonstrated that the addi-
tion of exogenous artiFcial RNA to the
cell-free translation system enhanced the
incorporation of particular amino acids
into polypeptides. The Frst demonstration
was that, in the presence of the polyur-
dylic acid (poly U), in
Escherichia coli
,the
cell-free translation system was capable of
synthesizing polymers of only phenylala-
nine at higher magnesium concentrations
than physiological conditions. This Fnding
in 1960 lead to the determination of the cor-
respondence of the triplet codon to amino
acid over the next Fve years. ±or instance,
it was immediately shown that poly C was
able to stimulate proline incorporation into
polypeptide, indicating that the codon CCC
corresponded to proline. By the various
synthesized RNAs consisting of random-
ized two or three kinds of nucleotides, the
stimulation of the incorporation of amino
acid into the polypeptide was examined,
and the relationship between codons and
amino acids was determined using these
polynucleotides. However, this approach
came to a standstill because statistical spec-
ulation resulted in ambiguity.
This problem was solved by the ele-
gant experiment called the
triplet-binding
experiment
, in which aminoacyl tRNA
charged with cognate amino acids binds
synthesized
trinucleotide on
ribosome
and determined the correspondence of
amino acids to triplet codons. Simultane-
ously, the chemically synthesized oligonu-
cleotide with a deFned sequence was
employed for examination of the incor-
poration of amino acid into polypeptides.
Together with these results, the genetic
code table had been established by 1966.
Another contribution of the cell-free
translation system was the direct veriF-
cation that tRNA is an adaptor molecule
linking amino acids to codons on mRNA.
The existence of an adaptor molecule
had been proposed by Crick, which had
not been evidenced with any experimen-
tal results. In 1957, Hoagland and Ogata
independently found that the small solu-
ble RNA molecule, which would later be
named tRNA, was charged with amino
acids. Nierenberg’s triplet-binding experi-
ment leads us to the conclusion that this
tRNA molecule plays the role of the adap-
tor molecule that deciphers comma-less
triplet codons on mRNA. The determi-
nation of the primary structure of tRNA
by Holley demonstrated that tRNA folded
in cloverleaf structure fulFlls the decod-
ing function based upon the interaction
between the anticodon and codon.
1.3
Elucidation of Factors Involved in the
Translation Process
To evaluate the role of protein factors
involved in the translation process, the cell-
free translation system has appeared to be
the most useful system. The translation
process proceeds in three steps: initiation,
elongation, and termination, in which
speciFc protein factors govern a respective
reFned process. These factors were Frst
discovered to enhance protein synthesis
using cell-free translation, and the detailed
behavior was elucidated by a well-deFned
system capable of verifying the elementary
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