The extent to which certain phage mutants could be reverted to wild type by different mutagens revealed a class of 'deletion' (minus) and 'insertion' (plus) mutants. Crosses between representatives of the two classes (plus/minus or minus/plus) would give wild-type progeny because the reading frame must have been restored, whereas crosses within a class (plus/plus or minus/minus) gave no wild-type progeny — the reading frame remained disrupted (showing, incidentally, that the code was not based on two or a multiple of two bases). The crucial insight came from the discovery that a triple mutant within either class (minus/minus/minus or plus/plus/plus) could give wild type: the frame had been restored. Hence the reading unit, or codon, must be three (or a multiple of three) bases.
In the 40 years since this dramatic discovery, the detailed mechanism of the decoding of the non-overlapping sequential triplets is still not understood, despite recent success in obtaining atomic-level structural information about the subunits of the ribosome. The ribosome is a gigantic complex of more than 50 proteins and RNA, the latter having the primal role in the decoding process. Understanding the dynamics between the ribosomal RNA, transfer RNA and messenger RNA (mRNA) that establish and maintain reading frames in the ribosome remains a challenge today.
