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Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro.
Open accessAbstract
A recombinant plasmid was constructed with six synthetic DNA oligomers such that the DNA sequence corresponding to yeast tRNA(Phe) is flanked by a T7 promoter and a BstNI restriction site. Runoff transcription of the BstNI-digested plasmid with T7 RNA polymerase gives an unmodified tRNA of the expected sequence having correct 5' and 3' termini. This tRNA(Phe) transcript can be specifically aminoacylated by yeast phenylalanyl-tRNA synthetase and has a Km only 4-fold higher than that of the native yeast tRNA(Phe). The Km is independent of Mg2+ concentration, whereas the Vmax is very dependent on Mg2+ concentration. Comparison of the melting profiles of the native and the unmodified tRNA(Phe) at different Mg2+ concentrations suggests that the unmodified tRNA(Phe) has a less stable tertiary structure. Using one additional DNA oligomer, a mutant plasmid was constructed having a guanosine to thymidine change at position 20 in the tRNA gene. A decrease in Vmax/Km by a factor of 14 for aminoacylation of the mutant tRNA(Phe) transcript is observed.
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Cited by
Karikó, K., Buckstein, M., Ni, H., & Weissman, D. (2005) · Immunity · doi:10.1016/j.immuni.2005.06.008 · Cited by 2,728
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Noren, C. J., Anthony-Cahill, S. J., Griffith, M. C., & Schultz, P. G. (1989) · Science · doi:10.1126/science.2649980 · Cited by 1,019
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Universal rules and idiosyncratic features in tRNA identity
Giege, R., Sissler, M., & Florentz, C. (1998) · Nucleic Acids Research · doi:10.1093/nar/26.22.5017 · Cited by 769
Metadata
- Authors
- Sampson, J. R., & Uhlenbeck, O. C.
- Venue
- Proceedings of the National Academy of Sciences
- Published
- 1988-02
- Volume
- 85
- Issue
- 4
- Pages
- 1033-1037
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- OpenAlex
- W2127749120
- Cited by
- 604
- References
- 0
- FWCI
- 11.05
- Type
- journal article
- Language
- en
- Open access
- green
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- Sampson, J. R., & Uhlenbeck, O. C. (1988). Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro. Proceedings of the National Academy of Sciences. https://doi.org/10.1073/pnas.85.4.1033
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