SELECTION FOR ACTIVE ESCHERICHIA-COLI TRANSFER RNA(PHE) VARIANTS FROMA RANDOMIZED LIBRARY USING 2 PROTEINS

In vitro selection was used to isolate active Escherichia coli tRNA(Ph e) variants from randomized libraries. Functional tRNAs were first sel ected by multiple rounds of binding to Escherichia coli phenylalanyl-t RNA synthetase. These variants were then aminoacylated and selected fo r affinity to elongation factor-Tu. By randomizing potential recogniti on nucleotides, the importance of residues U20, G34, A35, A36 and U59, previously identified to be required for specific recognition by E.co li phenylatanyl-tRNA synthetase (FRS), was confirmed. However, the seq uences of several active variants imply that the wild-type tertiary in teractions G10-C25-U45 and A26-G44 are not required for recognition, a s previously suggested. Selection of functional tRNAs from a second li brary randomized at positions normally involved in conserved tertiary interactions revealed new combinations-of nucleotides at these positio ns, suggesting the presence of novel tertiary interactions. In both li braries, active sequences containing deletions were isolated. Taken to gether, it is clear that FRS is active with substrates having an unexp ectedly broad sequence diversity. Finally, the potency of this method is illustrated by the identification of a second class of variants tha t was isolated by virtue of the presence of an impurity in the FRS pre paration.