The use of chemical nucleases to analyze RNA-protein interactions. The TFIIIA-5 S rRNA complex

Abstract

The complex of Xenopus transcription factor IIIA (TFIIIA) with 5 S rRNA was analyzed in nuclease protection experiments using hydroxyl radical. The protection pattern reveals that TFIIIA interacts with a substantial amount of the RNA molecule, with close association between the factor and the arm of the RNA composed of helix IV-loop E-helix V. Additional sites of protection punctuate the other arm of the molecule. Important contact sites within the complex were identified in "missing nucleoside" experiments. Random single-nucleoside gaps were introduced into 5 S rRNA using either Fe[EDTA]2-/H2O2 or bis(1,10-phenanthroline)copper(I). This modified RNA was allowed to bind to TFIIIA in an exchange reaction, and, afterward, bound and unbound fractions were separated on nondenaturing polyacrylamide gels. Missing nucleoside positions specifically enriched in the unbound fraction of RNA are located in the two strands that comprise loop E. These are not necessarily sites of sequence-specific contacts, but rather may constitute a region of secondary structure essential to recognition and binding of TFIIIA to 5 S rRNA.