The sequence of a coding strand of DNA, read in the direction from 5 to 3, consists of nucleotide triplets (codons) corresponding to the amino acid sequence of a protein read from N-terminus to C-terminus. Sequencing of DNA and proteins makes it possible to compare corresponding nucleotide and amino acid sequences directly. There are 64 codons (each of 4 possible nucleotides can occupy each of the three positions of the codon, making 43 = 64 possible trinucleotide sequences). Each of these codons has a specific meaning in protein synthesis: 61 codons represent amino acids; 3 codons cause the termination of protein synthesis.
The meaning of a codon that represents an amino acid is determined by the tRNA that corresponds to it; the meaning of the termination codons is determined directly by protein factors.
The breaking of the genetic code originally showed that genetic information is stored in the form of nucleotide triplets, but did not reveal how each codon specifies its corresponding amino acid. Before the advent of sequencing, codon assignments were deduced on the basis of two types of in vitro studies. A system involving the translation of synthetic polynucleotides was introduced in 1961, when Nirenberg showed that polyuridylic acid [poly(U)] directs the assembly of phenylalanine into polyphenylalanine. This result means that UUU must be a codon for phenylalanine. A second system was later introduced in which a trinucleotide was used to mimic a codon, thus causing the corresponding aminoacyl-tRNA to bind to a ribosome. By identifying the amino acid component of the aminoacyl-tRNA, the meaning of the codon can be found. The two techniques together assigned meaning to all of the codons that represent amino acids (Nirenberg and Matthaei, 1961; Nirenberg and Leder, 1964).
61 of the 64 codons represent amino acids. The other three cause termination of protein synthesis. The assignment of amino acids to codons is not random, but shows relationships in which the third base has less effect on codon meaning; also related amino acids are often represented by related codons.