Page 115 - Genetics_From_Genes_to_Genomes_6th_FULL_Part2

274 Chapter 8 Gene Expression: The Flow of Information from DNA to RNA to Protein

(see Chapter 7). Because point mutations that change only function by one mutation canceling another in the same
a single nucleotide pair affect only a single amino acid in a gene is known as intragenic suppression.
polypeptide, each nucleotide in a gene must influence the Crick and Brenner supposed not only that each codon
identity of only a single amino acid. In contrast, if a nucle­ is a trio of nucleotides, but that each gene has a single start­
otide were part of more than one codon, a mutation in that ing point. This starting point establishes a reading frame:
nucleotide would affect more than one amino acid. the sequential partitioning of nucleotides into groups of
three to generate the correct order of amino acids in the
resulting polypeptide chain (Fig. 8.4a). Changes that alter
Nonoverlapping Triplet Codons the grouping of nucleotides into codons are called
frameshift mutations; they shift the reading frame for all
Are Set in a Reading Frame codons beyond the point of insertion or deletion, almost

Although the most efficient code to specify 20 amino ac­ always abolishing the function of the polypeptide product.
ids requires three nucleotides per codon, more compli­ If codons are read in order from a fixed starting point,
cated scenarios are possible. But in 1955, Francis Crick a deletion (−) can counterbalance an insertion (+) to re­
and Sydney Brenner obtained convincing evidence for the store the reading frame (Fig. 8.4a). Note that the gene
triplet nature of the genetic code in studies of mutations in would regain its wild­type activity only if the portion of
the bacteriophage T4 rIIB gene originally characterized the polypeptide encoded between the two mutations of op­
by Seymour Benzer (Chapter 7). They induced the muta­ posite sign is not required for protein function, because in
tions with proflavin, an intercalating mutagen that can the double mutant, this region would have an improper
insert itself between the paired bases stacked in the center amino acid sequence. Also, the incorrect amino acids must
of the DNA molecule (recall Fig. 7.14c). Crick and not prevent the protein from folding into a functional
Brenner’s original assumption was that proflavin would conformation.
act like other mutagens, causing single­base substitutions. Crick and Brenner realized that they could use + and
If this were true, it would be possible to generate rever­ − mutations in rIIB to test the hypothesis that codons were
tants through treatment with other mutagens that might indeed nucleotide triplets. If codons are composed of three
−
restore the wild­type DNA sequence. nucleotides, then combining two different rIIB mutations
Surprisingly, genes with proflavin­induced mutations of the same sign (+ + or − −) in the same gene should
+
did not revert to wild­type upon treatment with other muta­ never lead to intragenic suppression (an rIIB phenotype).
gens known to cause nucleotide substitutions. Only further Combinations of three + or three − mutations, however,
+
exposure to proflavin caused proflavin­induced mutations should sometimes result in an rIIB revertant. These pre­
to revert to wild­type. Crick and Brenner had to explain this dictions were exactly verified by the results (Fig. 8.4c).
observation before they could proceed with their phage ex­
periments. With keen insight, they correctly guessed that Evidence that most amino acids are specified
proflavin does not cause base substitutions; instead, it
causes insertions or deletions of a single base pair. This by more than one codon
hypothesis explained why base­substituting mutagens As Fig. 8.4c illustrates, intragenic suppression occurs only
could not cause reversion of proflavin­induced mutations. if, in the region between two frameshift mutations of op­
posite sign, a gene still dictates the appearance of amino
acids—even if these amino acids are not the same as those
Evidence for a triplet code appearing in the normal protein. If the frameshifted part of
Crick and Brenner began their experiments with a particu­ the gene instead encodes instructions to stop protein syn­
−
lar proflavin­induced rIIB mutation they called FC0. thesis by introducing a triplet that does not correspond to
They next treated this mutant strain with more proflavin to any amino acid, then production of a functional polypep­
+
isolate an rIIB revertant (Fig. 8.4a). By recombining this tide will not be possible. The reason is that polypeptide
revertant with wild­type bacteriophage T4, Crick and synthesis would stop before the compensating mutation
Brenner were able to show that the revertant’s chromo­ could re­establish the correct reading frame.
−
some actually contained two different rIIB mutations The fact that intragenic suppression occurs as often as
(Fig. 8.4b). One was the original FC0 mutation; the other it does suggests that the code includes more than one codon
was the newly induced FC7. Either mutation by itself for some amino acids. Recall that there are 20 common
3
yields a mutant phenotype, but their simultaneous occur­ amino acids but 4 = 64 different combinations of three
+
rence in the same gene yielded an rIIB phenotype. Crick nucleotides. If each amino acid corresponded to only a sin­
and Brenner reasoned that if the first mutation was the gle codon, there would be 64 − 20 = 44 possible triplets
addition of a single base pair, represented by the symbol not encoding an amino acid. These noncoding triplets
(+), then the counteracting mutation must be the deletion would act as stop signals and prevent further polypeptide
of a base pair, represented as (−). The restoration of gene synthesis. In this scenario, more than half of all frameshift