PROBING OF TERTIARY INTERACTIONS IN RNA - 2'-HYDROXYL-BASE CONTACTS BETWEEN THE RNASE P RNA AND PRE-TRNA

摘要

A general method has been developed to analyze all 2' hydroxyl groups invoiced in tertiary interactions in RNA in a single experiment, This method involves comparing the activity of populations of circularly permuted RNAs that contain or lack potential hydrogen-bond donors at each position, The 2' hydroxyls of the pre-tRNA substrate identified as potential hydrogen bond donors in intermolecular interactions with the ribozyme from eubacterial RNase P (P RNA) are located in the T stem and T loop, acceptor stem, and 3' CCA regions, To locate the hydrogen-bond accepters for one of these 2' hydroxyls in the P RNA, a phylogenetically conserved adenosine was mutated to a guanosine. When this mutant P RNA was used, increased cleavage activity of a single circularly permuted substrate within the population was observed. The cleavage efficiency (k(cat)/K-m) of a singly 2'-deoxy-substituted substrate at this position in the T stem was also determined, For the wild-type P RNA, the catalytic efficiency was significantly decreased compared with that of the all-ribo substrate, consistent with the notion that this 2' hydroxyl plays an important role. For the P RNA mutant, no additional effect was found upon 2'-deoxy substitution, We propose that this particular 2' hydroxyl in the pre-tRNA interacts specifically with this adenosine in the P RNA. This method should be useful in examining the role of 2' hydroxyl groups in other RNA-RNA and RNA-protein complexes.