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Nucleic Acids Research, 1990, Vol. 18, No. 1 83-88
© 1990

MOLECULAR BIOLOGY

In vitro suppression of an amber mutation by a chemically aminoacylated transfer RNA prepared by runoff transcription

Christopher J. Noren, Spencer J. Anthony-Cahill, Karen A. Noren, Michael C. Griffith and Peter G. Schultz*

Department of Chemistry, University of California Berkeley, CA 94720, USA

* To whom correspondence should be addressed

Received October 11, 1989. Accepted November 27, 1989.

An amber suppressor tRNA was prepared in vitro by runoff transcription with T7 RNA polymerase. Both fulllength tRNA and truncated tRNA lacking the 3' terminal pCpA from the acceptor stem could be synthesized from the same DNA template. Truncated runoff suppressor tRNA could be enzymatically ligated to phenylalanyl-pCpA to generate aminoacylated fulllength suppressor tRNA (Phe-tRNACUA). Phe-tRNACUA is capable of suppressing an amber (UAG) mutation in vitro with equivalent efficiency as suppressor prepared by anticodon-loop replacement of a naturally-isolated tRNA. The ease of suppressor tRNA preparation using this method, compared to anticodon-loop replacement, greatly facilitates the use of chemically acylated suppressor tRNA's for site-specifically incorporating unnatural amino acids into proteins.


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