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Construction of Two Escherichia coli Amber Suppressor Genes: tRNACUAPhe and tRNACUACys

Normanly, Jennifer and Masson, Jean-Michel and Kleina, Lynn G. and Abelson, John and Miller, Jeffrey H. (1986) Construction of Two Escherichia coli Amber Suppressor Genes: tRNACUAPhe and tRNACUACys. Proceedings of the National Academy of Sciences of the United States of America, 83 (17). pp. 6548-6552. ISSN 0027-8424. PMCID PMC386541. doi:10.1073/pnas.83.17.6548.

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Amber suppressor genes corresponding to Escherichia coli tRNAPhe and tRNACys have been constructed for use in amino acid substitution studies as well as protein engineering. The genes for either tRNAGAAPhe or tRNAGCACys both with the anticodon 5' CTA 3' were assembled from four to six oligonucleotides, which were annealed and ligated into a vector. The suppressor genes are expressed constitutively from a synthetic promoter, derived from the promoter sequence of the E. coli lipoprotein gene. The tRNAPhe suppressor (tRNACUAPhe) is 54-100% efficient in vivo, while the tRNACys suppressor (tRNACUACys) is 17-50% efficient. To verify that the suppressors insert the predicted amino acids, both genes were used to suppress an amber mutation in a protein coding sequence. NH2-terminal sequence analysis of the resultant proteins revealed that tRNACUAPhe and tRNACUACys insert phenylalanine and cysteine, respectively. To demonstrate the potential of these suppressors, tRNACUAPhe and tRNACUACys have been used to effect amino acid substitutions at specific sites in the E. coli lac repressor.

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Additional Information:© 1986 by the National Academy of Sciences. Contributed by John Abelson, May 16, 1986. We thank Lynn Williams of the University of Southern California Microchemical Core Laboratory and Audree Fowler of University of California at Los Angeles for protein sequencing. The manuscript was improved by comments from V. Bankaitis, M. Clark, E. Grayhack, A. Lustig, E. Phizicky, and S. Ruby. This work was supported in part by a National Science Foundation grant (DMB-8417353) to J.A. and J.H.M., a National Research Service Award (T32GM07616) from the National Institute of General Medical Sciences to J.N., and a North Atlantic Treaty Organization fellowship and Philippe Foundation fellowship to J.-M.M. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Funding AgencyGrant Number
NIH Predoctoral FellowshipT32GM07616
North Atlantic Treaty Organization (NATO)UNSPECIFIED
Philippe FoundationUNSPECIFIED
Subject Keywords:gene synthesis; amino acid substitution; nonsense suppression
Issue or Number:17
PubMed Central ID:PMC386541
Record Number:CaltechAUTHORS:NORpnas86
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:5781
Deposited By: Archive Administrator
Deposited On:01 Nov 2006
Last Modified:08 Nov 2021 20:28

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