The other player in the translation process that we have not yet considered is transfer RNA, or tRNA. As we shall see, tRNA serves as an adaptor or intermediary between mRNA and amino acids. tRNAs are among the best characterized RNA molecules - they are quite short (75 to 90 nucleotides long) and have nearly identical sequences in ekaryotes and prokaryotes. tRNA molecules are somewhat unique in that they contain several unusual nucleotides, such as inosine, pseudouridine, and hypoxanthine.

The sequence of each individual tRNA molecule is such that base pairing occurs between strands in different regions of the same molecule. This gives tRNA molecules a characteristic 'cloverleaf' shape. There are two main functional regions of the tRNA molecule. The middle loop of the cloverleaf contains three unpaired bases known as the anticodon. The anticodon base pairs with the complementary codon on mRNA during translation. Directly opposite of the anticodon is a region with no loop - it contains both ends of the linear tRNA molecule. This region, particularly the 3' end of the tRNA is where a specific amino acid will bind in preparation for protein synthesis. A tRNA molecule with a particular anticodon sequence will only bind to one amino acid (for example, the tRNA with AGU as an anticodon sequence will only bind to the amino acid serine). In this way, specificity of the genetic code is maintained.

tRNA molecules are joined to their specific amino acid in a reaction known as charging. The 3' end of the tRNA molecule is covalently linked to the correct amino acid by an enzyme called aminoacyl tRNA synthetase. This enzyme recognizes the appropriate tRNA and enzyme, and uses the energy of ATP to join the two. Because the recognition of the tRNA and amino acid by the enzyme is so specific, there must be a different aminoacyl tRNA synthetase for each amino acid. Therefore, there are at least 20 different aminoacyl tRNA synthetases.