In Vitro Engineering Using Acyl-Derivatized tRNAs

  • Obed W. Odom
  • Wieslaw Kudlicki
  • Boyd Hardesty
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 77)

Abstract

Incorporation of fluorescently labeled or otherwise modified amino acids into proteins is potentially useful in a variety of ways, including monitoring the folding of the nascent protein chain, measuring posttranslational conformational changes and binding of substrates, and determining structure-function relationships. One of the most successful techniques for incorporating fluorescent amino acids into proteins has been to use aminoacyl-tRNAs in which the α-amino group has been acylated with a fluorescent moiety producing an N-acylaminoacyl-tRNA derivative (1). These derivatives have been used in then own right to study tRNA binding sites on the ribosome (2). Such derivatives, with their acylated amino groups, can only serve as donors in protein synthesis and thus can only be incorporated at the N-terminus. Nevertheless they have been very useful in studying the poly (U)1-directed synthesis of polyphenylalanine, polyserine, and polyalanine, the latter two using synthetic tRNAs whose anticodons had been changed to AAA (3). By incorporating coumarin-labeled phenylalanine or alanine at the N-terminus of such nascent chains, we were able to study by fluorescence, the interactions of the nascent chains with the ribosome as a function of chain length (4,5). More recently, using an efficient coupled transcription/translation system from Escherichia coli (6), we have incorporated coumarin-labeled methionine from tRNAf Met into several proteins. The fluorescent probe has been instrumental in studying interactions of those proteins with molecular chaperones (7).

Keywords

Cellulose Vortex Hydrolysis Sucrose Phenol 

References

  1. 1.
    Odom, O. W., Picking, W. D., and Hardesty, G. (1990) The movement of tRNA but not nascent peptide during peptide bond formation on ribosomes. Biochemistry 29, 10,734–10,744.PubMedCrossRefGoogle Scholar
  2. 2.
    Odom, O. W. and Hardesty, B. (1992) Use of 50 S-binding antibiotics to characterize the ribosomal sue to which peptidyl-tRNA is bound. J. Biol. Chem. 267, 19,117–19,122.PubMedGoogle Scholar
  3. 3.
    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.PubMedCrossRefGoogle Scholar
  4. 4.
    Picking, W. D., Odom, O. W., Tsalkova, T., Serdyuk, I., and Hardesty, B. (1991) The conformation of nascent polylysine and polyphenylalanine peptides on ribosomes. J. Biol. Chem. 266, 1534–1542.PubMedGoogle Scholar
  5. 5.
    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.PubMedCrossRefGoogle Scholar
  6. 6.
    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.PubMedCrossRefGoogle Scholar
  7. 7.
    Kudlicki, W., Odom, O. W., Kramer, G., and Hardesty, B. (1994) Chaperone-dependent folding and activation of ribosome-bound nascent rhodanese analysis by fluorescence. J. Mol. Biol. 244, 319–331.PubMedCrossRefGoogle Scholar
  8. 8.
    Rappaport, S. and Lapidot, Y. (1974) The chemical preparation of acetyl-aminoacyl-tRNA. Methods Enzymol 29, 685–688.CrossRefGoogle Scholar
  9. 9.
    Kuechler, E. and Barta, A. (1977) Aromatic derivatives of aminoacyl-tRNA as photoaffinity labels for ribosomes. Methods Enzymol. 46, 676–683.PubMedCrossRefGoogle Scholar
  10. 10.
    Johnson, A. E., Woodward, W. R., Herbert, E., and Menninger, J. R. (1976) Nε-Acetyllysine transfer ribonucleic acid: a biologically active analogue of aminoacyl transfer ribonucleic acid. Biochemistry 15, 569–575.PubMedCrossRefGoogle Scholar
  11. 11.
    Anderson, G. W., Zimmerman, J. E., and Callihan, F. M. (1964) The use of esters of N-hydroxysuccinimide in peptide synthesis. J. Am. Chem. Sot. 86, 1839–1842.CrossRefGoogle Scholar
  12. 12.
    Zubay, G. (1973) In vitro synthesis of protein in microbial systems. Annu. Rev. Genet. 7, 267–287.PubMedCrossRefGoogle Scholar
  13. 13.
    Adams, J. M. (1968) On the release of the formyl group from nascent protein. J. Mol. Biol. 33, 571–589.PubMedCrossRefGoogle Scholar
  14. 14.
    Miller, C. G., Strauch, K. L., Kukral, A. M., Miller, J. L., Wingfield, P. T., Mazei, G. J., Werlen, R. C., Graber, P., and Rao Movva, N. (1987) N-terminal methionine-specific peptidase in Salmonella typhimurium. Proc. Natl. Acad. Sci. USA 84, 2718–2722.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1998

Authors and Affiliations

  • Obed W. Odom
    • 1
  • Wieslaw Kudlicki
    • 1
  • Boyd Hardesty
    • 1
  1. 1.Department of Chemistry and BiochemistryUniversity of TexasAustin

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