In Vitro Selection of Small RNAs that Bind to Escherichia coli Phenylalanyl-tRNA Synthetase

Abstract

Small RNAs were selected from a highly degenerate library on the basis of their ability to bind tightly to Escherichia coli phenylalanyl-tRNA synthetase (FRS). The 63 nucleotide library consisted of the acceptor stem and portions of the D and T stems of E. coli tRNAPhe flanking a 32 nucleotide randomized region. Because FRS binding relies on a correctly folded tRNA substrate, the selected variants from this library were expected to resemble tRNAPhe structure. After seven cycles of selection, the RNA library bound to FRS with similar affinity to that of the E. coli tRNAPhe, but did not show detectable aminoacylation. Fourteen FRS-specific isolates were sequenced and found to contain an anticodon stem-loop including the anticodon triplet of tRNAPhe. The tight-binding RNAs fell into two classes depending on the location of this step-loop within the sequence. The acceptor stem defined by the non-randomized sequence was also found to be essential for binding Mutation of two residues within a common hexanucleotide sequence present in one of the classes reduced binding to FRS. Taken together, these results suggest that in order to bind RNAs tightly, FRS requires the simultaneous interaction of the anticodon stem-loop and acceptor stem, and additional sequences needed for proper folding. This approach should assist in the detection of motifs that resemble tRNA, but are too dissimilar to be identified by sequence comparison.