Quadruplet codons: implications for code expansion and the specification of translation step size

One of the requirements for engineering expansion of the genetic code is a unique codon which is available for specifying the new amino acid. The potential of the quadruplet UAGA in Escherichia coli to specify a single amino acid residue in the presence of a mutant tRNA(Leu) molecule containing the extra nucleotide, U, at position 33.5 of its anticodon loop has been examined. With this mRNA-tRNA combination and at least partial inactivation of release factor 1, the UAGA quadruplet specifies a leucine residue with an efficiency of 13 to 26 %. The decoding properties of tRNA(Leu) with U at position 33.5 of its eight-membered anticodon loop, and a counterpart with A at position 33.5, strongly suggest that in both cases their anticodon loop bases stack in alternative conformations. The identity of the codon immediately 5' of the UAGA quadruplet influences the efficiency of quadruplet translation via the properties of its cognate tRNA. When there is the potential for the anticodon of this tRNA to dissociate from pairing with its codon and to re-pair to mRNA at a nearby 3' closely matched codon, the efficiency of quadruplet translation at UAGA is reduced. Evidence is presented which suggests that when there is a purine base at position 32 of this 5' flanking tRNA, it influences decoding of the UAGA quadruplet.