Recognition of the T Stem−Loop of a Pre-tRNA Substrate by the Ribozyme from Bacillus subtilis Ribonuclease P

Abstract

The ribozyme from bacterial ribonuclease P (denoted P RNA) specifically recognizes the coaxially stacked T stem−loop and the acceptor stem of a tRNA substrate. This recognition is mediated primarily through tertiary interactions. At least four 2‘-OH groups in the T stem−loop region have been implicated as direct contacts with Bacillus subtilis P RNA [Pan, T., et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 12510]. Effects of six single 2‘-OH → 2‘-H substitutions and two base mutants of the G19−C56 tertiary interaction in tRNA on substrate binding (K_d) and the chemical step of the reaction (k₂) have been determined using a tRNA^Phe substrate containing a 2‘-deoxy residue at the cleavage site. Our results show that at least five functional groups in the T stem−loop of tRNA directly participate in P RNA binding. They include the 2‘-OH groups of residues 54, 56, 61, and 62 and possibly the 4-amino group of the conserved C56. The 2‘-OHs of residues 54, 61, and 62 are positioned within the same minor groove due to stacking of the reverse Hoogsteen U54-A58 pair on the G53-C61 Watson−Crick pair in the T stem. This groove is extended to the 4-amino group of C56 through the tertiary structure of tRNA. We use the term “tertiary groove” to describe alignment of functional groups through tertiary folding of an RNA. The binding also includes the 2‘-OH of nucleotide C56 which is not located in this tertiary groove. Assuming additivity, these five interactions can contribute 7.4 kcal/mol or 10⁵-fold in binding but only −0.5 kcal/mol or ∼2-fold in chemistry at 37 °C. The P RNA binding site for the T stem−loop includes at least the previously identified A230 as well as the A130 in B. subtilis P RNA. The K_d and k₂ data from the A130G mutant of B. subtilis P RNA suggest that A130 may be proximal to residue 56 in tRNA. These results show how the highly structured T stem−loop region in a pre-tRNA substrate is bound by the B. subtilis P RNA. This is among the first examples of how a nonhelical RNA structure can be recognized by another RNA through tertiary interactions.