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 pote ntial of the quadruplet UAGA in Escherichia roil 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 examin ed. With this mRNA-tRNA combination and at least partial inactivation of re lease factor 1, the UAGA quadruplet specifies a leucine residue with an eff iciency of 13 tr, 26 %. The decoding properties of tRNA(Leu) with U at posi tion 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 ba ses stack in alternative conformations. The identity of the codon immediate ly 5' of the UAGA quadruplet influences the efficiency of quadruplet transl ation via the properties of its cognate tRNA. When there is the potential f or 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 sug gests that when there is a purine base at position 32 of this 5' flanking t RNA, it influences decoding of the UAGA quadruplet. (C) 2000 Academic Press .