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 mutations by means of an extended anticodon loop, and a few have been used in protein mutagenesis. In an effort to expand the number of possible ways to uniquely and efficiently encode unnatural amino acids, we have devised a general 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 selectants included both known and novel suppressible four-base codons and resulted in a set of very efficient, non-cross-reactive tRNA/four-base codon pairs for AGGA, UAGA, CCCU and CUAG. The most efficient four-base codon suppressors had Watson-Crick complementary anticodons, and the sequences of the anticodon loops outside of the anticodons varied with the anticodon. Additionally, four-base codon reporter libraries were used to identify "shifty" sites at which +1 frameshifting is most favorable in the absence of suppressor tRNAs in Escherichia coli. We intend to use these tRNAs to explore the limits of unnatural polypeptide biosynthesis, both in vitro and eventually in vivo. In addition, this selection strategy is being extended to identify novel five- and six-base codon suppressors.
Magliery
T J
TJ
Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA.
Anderson
J C
JC
Schultz
P G
PG
D000595:Amino Acid Sequence; D000667:Ampicillin / Q000494:pharmacology; D000926:Anticodon / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020029:Base Pairing; D001483:Base Sequence; D002511:Cephalosporins / Q000378:metabolism; D003062:Codon / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D016368:Frameshift Mutation / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D015723:Gene Library; D017930:Genes, Reporter / Q000235:genetics; D005815:Genetic Code / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D014176:Protein Biosynthesis / Q000187:drug effects / Q000235:genetics; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012362:RNA, Transfer, Ser / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013489:Suppression, Genetic / Q000235:genetics*; D001618:beta-Lactamases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D000926:Anticodon; D002511:Cephalosporins; D003062:Codon; D012362:RNA, Transfer, Ser; D012694:Serine; D000667:Ampicillin; D012343:RNA, Transfer; D001618:beta-Lactamases; C021720:nitrocefin