Essential Cell Biology 5th edition

From RNA to Protein
247
5’
UUUUUUUUUUUUUUUUUUUUUUUU
synthetic mRNA
3’
cell-free translation
system plus radioactive
amino acids
N
Phe Phe Phe Phe Phe Phe Phe Phe
radioactive polypeptide synthesized
C
Figure 7–29 UUU codes for
phenylalanine. Synthetic
mRNAs are fed into a cell-free
translation system containing
bacterial ribosomes, tRNAs,
enzymes, and other small
molecules. Radioactive amino
acids were added to this mix,
one per experiment; when
the “correct” amino acid was
added, a radioactive polypeptide
would be produced. In this case,
poly U is shown to encode a
polypeptide containing only
phenylalanine.
polyribonucleotides. He found that if he started out by
making DNAs of a defined sequence, he could then use
RNA polymerase to produce RNAs from those. In this
way, Khorana prepared a collection of different RNAs
of defined repeating sequence: he generated ECB5 e7.27/7.29 sequences
of repeating dinucleotides (such as poly UC), trinucleotides
(such as poly UUC), or tetranucleotides (such as
poly UAUC).
These mixed polynucleotides, however, yielded results
that were much more difficult to decode than the mononucleotide
messages that Nirenberg had used. Take poly
UG, for example. When this repeating dinucleotide was
added to the translation system, researchers discovered
that it codes for a polypeptide of alternating cysteines
and valines. The RNA, of course, contains two different,
alternating codons: UGU and GUG. So the researchers
could say that UGU and GUG code for cysteine and
valine, although they could not tell which went with
which. Thus these mixed messages provided useful
information, but they did not definitively reveal which
codons specified which amino acids (Figure 7–30).
Trapping the triplets
These final ambiguities in the code were resolved when
Nirenberg and a young medical graduate named Phil
Leder discovered that RNA fragments that were only
three nucleotides in length—the size of a single codon—
could bind to a ribosome and attract the appropriate
amino-acid-containing tRNA molecule. These complexes—containing
one ribosome, one mRNA codon,
and one radiolabeled aminoacyl-tRNA—could then be
captured on a piece of filter paper and the attached
amino acid identified.
Their trial run with UUU—the first word—worked splendidly.
Leder and Nirenberg primed the usual cell-free
translation system with snippets of UUU. These trinucleotides
bound to the ribosomes, and Phe-tRNAs
bound to the UUU. The new system was up and running,
and the researchers had confirmed that UUU codes for
phenylalanine.
All that remained was for researchers to produce all 64
possible codons—a task that was quickly accomplished
in both Nirenberg’s and Khorana’s laboratories. Because
these small trinucleotides were much simpler to synthesize
chemically, and the triplet-trapping tests were
easier to perform and analyze than the previous decoding
experiments, the researchers were able to work out
the complete genetic code within the next year.
MESSAGE
PEPTIDES
PRODUCED
poly UG ...Cys–Val–Cys–Val... UGU
GUG
poly AG
poly UUC
poly UAUC
...Arg–Glu–Arg–Glu...
...Phe–Phe–Phe...
+
...Ser–Ser–Ser...
+
...Leu–Leu–Leu...
...Tyr–Leu–Ser–Ile...
CODON
ASSIGNMENTS
AGA
GAG
UUC
UCU
CUU
UAU
CUA
UCU
AUC
Cys, Val*
Arg, Glu
Phe, Ser,
Leu
Tyr, Leu,
Ser, Ile
* One codon specifies Cys, the other Val, but which is which?
The same ambiguity exists for the other codon assignments
shown here.
Figure 7–30 Using synthetic RNAs of mixed, repeating
ribonucleotide sequences, scientists further narrowed
the coding possibilities. Because these mixed messages
produced mixed polypeptides, they did not permit the
unambiguous assignment of a single codon to a specific amino
acid. For example, the results of the poly-UG experiment
cannot distinguish whether UGU or GUG encodes cysteine.
As indicated, the same type of ambiguity confounded the
interpretation of all the ECB5 experiments e7.28/7.30 using di-, tri-, and
tetranucleotides.