Abstract
Synthetic tRNAs—those that are transcribed in vitro from a DNA sequence that has been ligated into an appropriate plasmid—have a wide variety of applications that range from testing tRNAs for their requirements for amino-acylation (1) to providing reagents for the investigation of protein folding (2). The examples that will be described here involve specific tRNAs with altered anticodons. The construction of several of these tRNAs will be given in detail as examples of the procedure applied. Also, methods of use and how to test the efficiency of these tRNAs will be reviewed.
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References
Hall, K. B., Sampson, J. R., Uhlenbeck, O. C., and Redfield, A. G. (1989) Structure of an unmodified tRNA molecule. Biochemistry 28, 5794–5801.
Picking, W. D., Picking, W. L., Odom, O. W., and Hardesty, B. (1992) Fluorescence characterization of the environment encountered by nascent polyalanine and polyserine as they exit Escherichia coli ribosomes during translation. Biochemistry 31, 2368–2375.
McClain, W. H. (1993) Rules that govern tRNA identity in protein syntheses. J. Mol. Biol. 234, 257–280.
Saks, M. E., Sampson, J. R., and Abelson, J. N. (1994) The tRNA identity problem, a search for rules. Science 263, 191–197.
Picking, W., Picking, W. D., and Hardesty, B. (1991) The use of synthetic tRNAs as probes for examining nascent peptides on Escherichia coli ribosomes. Biochimie 73, 1101–1107.
Picking, W. L., Picking, W. D., Ma, C., and Hardesty, B. (1991) A synthetic alanyl-initiator tRNA with initiator tRNA properties as determined by fluorescence measurements: comparison to a synthetic alanyl-elongator tRNA. Nucleic Acids Res. 19, 5749–5754.
Ma, C., Kudlicki, W., Odom, O. W., Kramer, G., and Hardesty, G. (1992) In vitro protein engineering using synthetic tRNAAla with different anticodons. Biochemistry 32, 7939–7945.
Wada, K., Aota, S., Tsuchtya, R., Ishibasht, F., Gojobori, T., and Ikemura, T. (1990) Codon usage tabulated from the GenBank genetic sequence data. Nucleic Acids Res. 18, 2367.
Ikemura, T. (1981) Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes. J. Mol. Biol. 146, 1–21.
Komine, Y., Adachi, T., Inokuchi, H., and Ozeki, H. (1990) Genomic organization and physical mapping of the transfer RNA genes in Escherichia coli K12. J. Mol. Biol. 212, 579–598.
Roth, J. R. (1981) Frameshift Suppression. Cell 24, 601, 602.
Tuohy, T. M. F., Thompson, S., Gesteland, R. F., and Atkins, J. F. (1992) Seven, eight and nine-membered anticodon loop mutants of tRNAArg-2 which cause +1 frameshifting. J. Mol. Biol. 228, 1042–1054.
Steinberg, S., Misch, A., and Sprinzl, M. (1993) Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res. 21, 3011–3015 (hardcopy published by Molekulargenetischer Arbeitskreis Rhein/Main e V., Bayreuth, Germany).
Sambrook, J., Fritsch, E. F., and Mamatis, T. (1989) Molecular Cloning: A Laboratory Manual 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Butler, E. T. and Chamberlin, M. J. (1982) Bacteriophase SP6-specific RNA polymerase. J. Biol. Chem. 257, 5772–5778.
Davanloo, P., Rosenberg, A. H., Dunn, J. J., and Studier, F. W. (1984) Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 81, 2035–2039.
Odom, O. W., Kudlicki, W., and Hardesty, B. (1997), In vitro engineering using acyl-derivatized tRNAs, in Protein Synthesis: Methods and Protocols (Martin, R., ed.) Humana, Totowa, NJ.
Mullis, K., Faloona, F., Scharf, S., Salki, R., Horn, G., and Erlich, H. (1989) in PCR Technology (Erlich, H., ed.), Stockton, NY, pp. 63–70.
Kudlicki, W., Kramer, G., and Hardesty, B. (1992) High efficiency cell-free synthesis of proteins refinement of the coupled transcription-translation system. Anal. Biochem. 206, 389–393.
Kudlicki, W., Odom, O. W., Kramer, G., and Hardesty, B. (1994) Activation and release of enzymatically inactive, full-length rhodanese that is bound to ribosomes as peptidyl-tRNA. J. Biol. Chem. 269, 16,549–16,553.
Baccanari, D., Phillips, A., Smith, S., Sinski, D., and Burchall, J. (1975) Purification and properties of Escherichia coli dihydrofolate reductase. Biochemistry 14, 5267–5273.
Schagger, H. and von Jagow, G. (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368–379.
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Kramer, G., Kudlicki, W., Hardesty, B. (1998). In Vitro Engineering Using Synthetic tRNAs with Altered Anticodons Including Four-Nucleotide Anticodons. In: Martin, R. (eds) Protein Synthesis. Methods in Molecular Biology, vol 77. Springer, Totowa, NJ. https://doi.org/10.1385/0-89603-397-X:105
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DOI: https://doi.org/10.1385/0-89603-397-X:105
Publisher Name: Springer, Totowa, NJ
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