Page 118 - Genetics_From_Genes_to_Genomes_6th_FULL_Part2

8.1 The Genetic Code 277

synthetic mRNAs only three nucleotides in length to an acid correspondences shown in the genetic code table
in vitro translational system containing tRNAs attached to (see Fig. 8.2).
amino acids, where only one of the 20 amino acids was
radioactive. They then poured through a filter a mixture of a
synthetic mRNA and the translational system containing a Polarities: 5′ to 3′ in mRNA corresponds
tRNA­attached, radioactively labeled amino acid (Fig. 8.6). to N to C in the polypeptide
tRNAs carrying an amino acid normally go right through In studies using synthetic mRNAs, when investigators
a filter. If, however, a tRNA carrying an amino acid binds added the six­nucleotide­long 5′ AAAUUU 3′ to an in vitro
to a ribosome, it will stick in the filter, because this larger translational system, the product N Lys­Phe C emerged,
complex of ribosome, amino­acid­carrying tRNA, and but no N Phe­Lys C appeared. Because AAA is the codon
small mRNA cannot pass through. for lysine and UUU is the codon for phenylalanine, this
Nirenberg and Leder used this approach to see which result means that the codon closest to the 5′ end of the
small mRNA caused the entrapment of which radioac­ mRNA encoded the amino acid closest to the N terminus of
tively labeled amino acid. For example, they knew from the corresponding polypeptide. Similarly, the codon near­
Khorana’s earlier work that CUC encoded either serine or est the 3′ end of the mRNA encoded the amino acid nearest
leucine. When they added the synthetic triplet CUC to an the C terminus of the resulting polypeptide.
in vitro system where the radioactive amino acid was ser­ To understand how the polarities of the macromole­
ine, this tRNA­attached amino acid passed through the cules participating in gene expression relate to each other,
filter, and the filter thus emitted no radiation (Fig. 8.6). remember that although the gene is a segment of a DNA
But when they added the same triplet to a system where double helix, only one of the two strands serves as a tem­
the radioactive amino acid was leucine, the filter lit up plate for the mRNA. This strand is known as the template
with radioactivity, indicating that the radioactively tagged strand. The other strand is the RNA-like strand, because
leucine attached to a tRNA had bound to the ribosome­mRNA it has the same polarity and sequence (written in the DNA
complex and gotten stuck in the filter (Fig. 8.6). CUC dialect) as the RNA. Note that some scientists use the terms
thus encodes leucine, not serine. Nirenberg and Leder sense strand or coding strand as synonyms for the RNA­
used this technique to determine most of the codon–amino like strand; in these alternative nomenclatures, the template
strand would be the antisense strand or the noncoding
strand. Figure 8.7 diagrams the respective polarities of a
Figure 8.6 Cracking the genetic code with mini-mRNAs. gene’s DNA, the mRNA transcript of that DNA, and the
Nirenberg and Leder added trinucleotides of known sequence, in resulting polypeptide.
combination with a mixture of amino acid–charged tRNAs where
only one amino acid was radioactive, to an in vitro extract
containing ribosomes. If the trinucleotide specified this amino acid, Nonsense codons and polypeptide
the radioactive charged tRNA formed a complex with the ribosomes
that could be trapped on a filter. The experiments shown here chain termination
indicate that the codon CUC specifies leucine, not serine. Although most of the simple, repetitive RNAs synthesized
14 C Ser 14 C Leu by Khorana were very long and thus generated very long
polypeptides, a few did not. These RNAs had signals that
C U C C U C stopped construction of a polypeptide chain. As it turned
+ 5' 3' + 5' 3'
out, three different triplets—UAA, UAG, and UGA—do
not correspond to any of the amino acids. When these
Labeled Ser tRNA + Labeled Leu tRNA +
Add ribosomes
synthetic trinucleotide synthetic trinucleotide
Figure 8.7 Correlation of polarities in DNA, mRNA, and
14
C Leu
polypeptide. The template strand of DNA is complementary to
both the RNA-like DNA strand and the mRNA. The 5′-to-3′ direction
Pour through in an mRNA corresponds to the N terminus-to-C terminus direction
filter Ribosome in the polypeptide.
RNA-like strand
C U C C U CC U C 5' 3'
DNA
No radioactivity Radioactivity 3' 5'
trapped in filter trapped in filter Template strand
14
C Ser
mRNA 5' 3'

Polypeptide N C