An RNA chaperone activity of non-specific RNA binding proteins in hammerhead ribozyme catalysis

Abstract

We have previously shown that a protein derived from the p7 nucleocapsid (NC) protein of HIV type-1 increases k(cat)/K(m) and k(cat) for cleavage of a cognate substrate by a hammerhead ribozyme. Here we show directly that the increase in k(cat)/K(m) arises from catalysis of the annealing of the RNA substrate to the ribozyme and the increase in k(cat) arises from catalysis of dissociation of the RNA products from the ribozyme. A peptide polymer derived from the consensus sequence of the C-terminal domain of the hnRNP A1 protein (A1 CTD) provides similar enhancements. Although these effects apparently arise from non-specific interactions, not all non-specific binding interactions led to these enhancements. NC and A1 CTD exert their effects by accelerating attainment of the thermodynamically most stable species throughout the ribozyme catalytic cycle. In addition, NC protein is shown to resolve a misfolded ribozyme-RNA complex that is otherwise long lived. These in vitro results suggest that non-specific RNA binding proteins such as NC and hnRNP proteins may have a biological role as RNA chaperones that prevent misfolding of RNAs and resolve RNAs that have misfolded, thereby ensuring that RNA is accessible for its biological functions.