Given the different numbers of “letters” in the mRNA and protein “alphabets,” scientists theorized that combinations of nucleotides corresponded to single amino acids. Nucleotide doublets would not be sufficient to specify every amino acid because there are only 16 possible two-nucleotide combinations (42). In contrast, there are 64 possible nucleotide triplets (43), which is far more than the number of amino acids. Scientists theorized that amino acids were encoded by nucleotide triplets and that the genetic code was degenerate. In other words, a given amino acid could be encoded by more than one nucleotide triplet. This was later confirmed experimentally; Francis Crick and Sydney Brenner used the chemical mutagen proflavin to insert one, two, or three nucleotides into the gene of a virus. When one or two nucleotides were inserted, protein synthesis was completely abolished. When three nucleotides were inserted, the protein was synthesized and functional. This demonstrated that three nucleotides specify each amino acid. These nucleotide triplets are called codons. The insertion of one or two nucleotides completely changed the triplet reading frame, thereby altering the message for every subsequent amino acid (Figure 1). Though insertion of three nucleotides caused an extra amino acid to be inserted during translation, the integrity of the rest of the protein was maintained.

Figure 1. The deletion of two nucleotides shifts the reading frame of an mRNA and changes the entire protein message, creating a nonfunctional protein or terminating protein synthesis altogether.

Scientists painstakingly solved the genetic code by translating synthetic mRNAs in vitro and sequencing the proteins they specified (Figure 2).

Figure 2. This figure shows the genetic code for translating each nucleotide triplet in mRNA into an amino acid or a termination signal in a nascent protein. (credit: modification of work by NIH)

In addition to instructing the addition of a specific amino acid to a polypeptide chain, three of the 64 codons terminate protein synthesis and release the polypeptide from the translation machinery. These triplets are called nonsense codons, or stop codons. Another codon, AUG, also has a special function. In addition to specifying the amino acid methionine, it also serves as the start codon to initiate translation. The reading frame for translation is set by the AUG start codon near the 5′ end of the mRNA. Following the start codon, the mRNA is read in groups of three until a stop codon is encountered.

The arrangement of the coding table reveals the structure of the code. There are sixteen “blocks” of codons, each specified by the first and second nucleotides of the codons within the block, e.g., the “AC*” block that corresponds to the amino acid threonine (Thr). Some blocks are divided into a pyrimidine half, in which the codon ends with U or C, and a purine half, in which the codon ends with A or G. Some amino acids get a whole block of four codons, like alanine (Ala), threonine (Thr) and proline (Pro). Some get the pyrimidine half of their block, like histidine (His) and asparagine (Asn). Others get the purine half of their block, like glutamate (Glu) and lysine (Lys). Note that some amino acids get a block and a half-block for a total of six codons.

The specification of a single amino acid by multiple similar codons is called “degeneracy.” Degeneracy is believed to be a cellular mechanism to reduce the negative impact of random mutations. Codons that specify the same amino acid typically only differ by one nucleotide. In addition, amino acids with chemically similar side chains are encoded by similar codons. For example, aspartate (Asp) and glutamate (Glu), which occupy the GA* block, are both negatively charged. This nuance of the genetic code ensures that a single-nucleotide substitution mutation might specify the same amino acid but have no effect or specify a similar amino acid, preventing the protein from being rendered completely nonfunctional.

The genetic code is universal. With a few exceptions, virtually all species use the same genetic code for protein synthesis. Conservation of codons means that a purified mRNA encoding the globin protein in horses could be transferred to a tulip cell, and the tulip would synthesize horse globin. Having only one genetic code is powerful evidence that all of life on Earth shares a common origin, especially considering that there are about 1084 possible combinations of 20 amino acids and 64 triplet codons.

Degeneracy is believed to be a cellular mechanism to reduce the negative impact of random mutations. Codons that specify the same amino acid typically only differ by one nucleotide. In addition, amino acids with chemically similar side chains are encoded by similar codons. This nuance of the genetic code ensures that a single-nucleotide substitution mutation might either specify the same amino acid but have no effect or specify a similar amino acid, preventing the protein from being rendered completely nonfunctional.