Expanding the genetic code: Selection of efficient suppressors of four-base codons and identification of "shifty" four-base codons with a library approach in Escherichia coli

Naturally occurring tRNA mutants are known that suppress +1 frameshift muta tions by means of an extended anticodon loop, and a few have been used in p rotein mutagenesis. In an effort to expand the number of possible ways to u niquely and efficiently encode unnatural amino acids, we have devised a gen eral strategy to select tRNAs with the ability to suppress four-base codons from a library of tRNAs with randomized 8 or 9 nt anticodon loops. Our sel ectants included both known and novel suppressible four-base codons and res ulted in a set of very efficient, non-cross-reactive tRNA/four-base codon p airs for AGGA, UAGA, CCCU and CUAG. The most efficient four-base codon supp ressors had Watson-Crick complementary anticodons, and the sequences of the anticodon loops outside of the anticodons varied with the anticodon. Addit ionally, four-base codon reporter libraries were used to identify "shifty" sites at which tl frameshifting is most favorable in the absence of suppres sor tRNAs in Escherichia coil. We intend to use these tRNAs to explore the limits of unnatural polypeptide biosynthesis, both in vitro and eventually ill vivo. In addition, this selection strategy is being extended to identif y novel five- and six-base codon suppressors. (C) 2001 Academic Press.