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The genetic code

As it became evident that genes controlled the structure of polypeptides, attention focused on how
the sequence of the four base pairs in DNA could control the sequence of the 20 amino acids found
in proteins. With the discovery of the mRNA intermediary, the question became one of how the
sequence of the four bases present in mRNA molecules could specify the amino acid sequence of a
polypeptide. What is the nature of the genetic code ralating mRNA base sequences (or DNA base-
pair sequences) to amino acid sequences? Clearly, the symbols or letters used in the code must be
the bases; but what comparises a codon the unit or word specifying amino acid (or, actually, one
aminoacyl-tRNA complex)?

Figure 10.29 Probable secondary structure of the human snRNA U1 and its potential base-pairing to
the 5 consensus sequence of introns in nuclear pre-mRNAs. The pre-mRNA sequence is shown in
red with the 5 consensus sequence (5 GUAAGU-3) of the intron written 3 to 5 to align it with the
complementary sequence near the 5 end of the U1 snRNA. The m symbols refer to methyl side
groups. The U1 snRNA has a trimethyl G cap at the 5 terminus linked to the subterminal Am by a 5-
5 triphosphate linkage like most eukaryotic mRNAs.

Three Nucleotides per Codon

Twenty different amino acids are incorporated during translation. Thus, at least 20 different codons
must be formed using the four symbols (bases) available in the message (mRNA). Two bases per
codon would yield only 16 possible codons Clearly not enough. Three bases per codon yield 64
possible codons- an apparent excess.

The first strong evidence that the genetic code was in fact a triplet code (three nucleotides per
codon) resulted from a genetic analysis of proflavin-induced mutations in the rII locus of phage T4
carried out by F. H. C Crick and colleagues in 1961. Crick and colleagues isolated proflavin-induced
revertants of a proflavin-induced mutation. (Proflavin, an acridine dye, induces single base-pair
additions and deletions). These revertants were shown to result from the occurrence of suppressor
mutations rather than from back-mutation at the original site of mutation. Crick and colleagues
reasoned that if the original mutation was a single base-pair addition or deletions or additions,
respectively, occuring at a site or sites near the original mutation. A single base-pair addition or
deletion will alter the reading frame of the gene nd mRNA (the codons in phase during translation).
When the suppressor mutations were isolated as a single mutants by screening progeny of
backrosses to wild type, they were found to produce mutant phenotypes, just like original mutation.
Crick and colleagues next isolated proflavin-induced suppressor mutations of the original suppressor
mutations, and so on.

All the isolated mutations were then classified into two groups, plus (+) and minus (-) (for additions
and deletios, although Crick had no idea which group was which) using the reasoning that a (+)
mutations would suppress a (-) mutation, but not another (+) mutation, and vice versa. Next, crick
constructed recombinants that carried various combinations of the (+) and the (-) mutations.
Recombinants with two (+) mutations or two (-) mutations always had mutant phenotypes, just like
the single mutants. Recombinants carrying three (-) mutations or three (-) mutations. However,
often had wild-type phenotypes. This indicated that the additions of three base-pairs or the deletion
of three base-pairs left the distal portion of the gene with the correct (wild-type) reading frame, a
result that would be expected only if each codon contained three nucleotides.

Confirmation that the coding ratio (nucleotides to amino acids) is indeed three has come from many
sources. Considerable evidence favoring a triplet code evolved from studies using in vitro translation
systems. The following observations were of major importance. (1) trinucleotides were found
sufficient to stimulate specific binding of aminoacyl-tRNAs to ribosomes. For example, 5 UUC- 3
stimulates ribosomalbinding of phenylalanyl tRN phe (2) chemically synthesized RNA molecules,
containing repeating dinucleotide sequences, directed the synthesis of copolymers with alternating
amino acids sequence. Poly (UG)n, for example, when used as an artificial mRNA in an in vitro
system, directed the synthesis of the repeating copolymer (cys-val)n (3) Molecules with repeating
trinucleotide sequences, on the other hand, directed the synthesis of a mixture of polyserine,
polyarginine, and polyvaline. Again, these results are only consistent with a triplet code. Ultimately,
the triplet nature of the code was definitively established by the results of correlated nucleic acid
and protein sequecing.

Deciphering the code

The deciphering of the genetic code that is, determining (1) which codons specify which amino
acids, (2) how many of the 64 possible codons are used, (3) how the code is punctuated, and (4)
whether different species use the same or different codons took place during the early 1960s and
was one of the most exciting periods in the history of science. The cracking of the genetic code
had an effect on the life sciences like the splitting of the atom did on the physical sciences. It opened
up a vast new field of study of gene expression.

The first major breakthrough came in 1961 when M.W.Nirenberg (1968 Nobel Prize recipient) and
J.H.Matthaei and then S. Ochoa (1959 Nobel Prize recipient) and coworkers demonstrated that
synthetic RNA molecules could be used as artificial mRNAs to direct in vitro protein synthesis. That
is, when ribosomes, aminoacyl-tRNAs, and the soluble protein 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 composition, the composition of the polypeptides synthesized 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)n]. Ochoa and others continued this approach using synthetic RNAs with random
sequences of known nucleotide composition, such as 50 percent U and 50 percent G. The
frequencies of the different triplets in such a random copolymer can be easily calculated. For
example, the 50 percent U/ 50 percent G copolymer will contain 12,5 percent of eight possible
codons: UUU, UUG, UGU, GUU, UGG, GUG, GGU, and GGG. These can then be compared with the
amino acids incorporated (phenylalanine, leucine, cysteine, valine, tryptophan, and glycine) when
this random copolymer is used in an in vitro protein-synthesizing system. By varying the
composition, for example, to 75 percent U and 25 percent G, one can vary the relative frequencies of
the eight codons and correlate them with the relative frequencies of the amino acids in the
polypeptides synthesized. Such experiments provided a great deal of information about the nature
of the code.
More definitive data were later obtained by H. G. Khorana using in vitro systems that were activated
by synthetic mRNAs of known nucleotide sequences. Khoranas experiments premitted direct
comparasions between nucleotide sequences and the amino acids incorporated in response to these
sequences. The ultimate cracking of the code occured when trinucleotides were found to function
as mini-mRNAs in directing the specific binding of aminoacyl-tRNAs to ribosomes. By using all the
64 possible trinucleotide sequences in such aminoacyl-tRNA binding experoments, it was possible to
verify the codon assignments made from data of earlier experiments.

Several lines of evidence now indicate that these codon assignments are correct for protein
synthesis in vitro for most, if not all, species. When the amino acid substitutions that result from
mutations induced with chemical mutagens with specific mutagenic effects are determined by
amino acid sequencing, the substitutions are almost always consistent with the codon assignments
and known the effect of the mutagen. More convincingly, when the nucleotide sequences of genes
or of mRNAs are determined and compared with the amino acid sequences of the polypeptides
encodedbby those genes or mRNAs, the observed correlations are always found to be those
predicted drom the accepted codon assignments. This can be illustrated by comparing the
nucleotide sequence of the gene coding for the protein coat or capsid of bacteriophage MS2 with
the amino acid sequence of the capsid polypeptide. Phage MS2 stores its genetic information in
RNA. Its chromosome is equivalent to an mRNA molecule in organisms with DNA genomes

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