Selection for active E.coli tRNAphe variants from a randomized library using two proteins

E. T. Peterson*, J. Blank, M. Sprinzl, O. C. Uhlenbeck

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

48 Scopus citations

Abstract

In vitro selection was used to isolate active Escherichia coli tRNAphe variants from randomized libraries. Functional tRNAs were first selected by multiple rounds of binding to Escherichia coli phenylalanyl-tRNA synthetase. These variants were then aminoacylated and selected for affinity to elongation factor-Tu. By randomizing potential recognition nucleotides, the importance of residues U20, G34, A35, A36 and U59, previously identified to be required for specific recognition by E.coli phenylalanyl-tRNA synthetase (FRS), was confirmed. However, the sequences of several active variants imply that the wild-type tertiary interactions G10-C25-U45 and A26-G44 are not required for recognition, as previously suggested. Selection of functional tRNAs from a second library randomized at positions normally involved in conserved tertiary interactions revealed new combinations of nucleotides at these positions, suggesting the presence of novel tertiary interactions. In both libraries, active sequences containing deletions were isolated. Taken together, it is dear that FRS is active with substrates having an unexpectedly broad sequence diversity. Finally, the potency of this method is illustrated by the identification of a second class of variants that was isolated by virtue of the presence of an impurity in the FRS preparation.

Original languageEnglish (US)
Pages (from-to)2959-2967
Number of pages9
JournalEMBO Journal
Volume12
Issue number7
StatePublished - 1993

Keywords

  • Aminoacyl-tRNA synthetase
  • Elongation factor-tu
  • Recognition
  • Selection
  • TRNA

ASJC Scopus subject areas

  • General Immunology and Microbiology
  • General Biochemistry, Genetics and Molecular Biology
  • Molecular Biology
  • General Neuroscience

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