RECOGNITION OF THE T-STEM-LOOP OF A PRE-TRANSFER-RNA SUBSTRATE BY THERIBOZYME FROM BACILLUS-SUBTILIS RIBONUCLEASE-P

The ribozyme from bacterial ribonuclease P (denoted P RNA) specificall y recognizes the coaxially stacked T stem-loop and the acceptor stem o f a tRNA substrate. This recognition is mediated primarily through ter tiary interactions. At least four 2'-OH groups in the T stem-loop regi on have been implicated as direct contacts with Bacillus subtilis P RN A [Pan, T., et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 12510]. Ef fects 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 (k(2)) have been determined usi ng 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-ami no group of the conserved C56. The 2'-OHs of residues 54, 61, and 62 a re positioned within the same minor groove due to stacking of the reve rse Hoogsteen U54-A58 pair on the G53-C61 Watson-Crick pair in the T s tem. This groove is extended to the 4-amino group of C56 through the t ertiary structure of tRNA. We use the term ''tertiary groove'' to desc ribe alignment of functional groups through tertiary folding of an RNA . The binding also includes the 2'-OH of nucleotide C56 which is not l ocated in this tertiary groove. Assuming additivity, these five intera ctions can contribute 7.4 kcal/mol or 10(5)-fold in binding but only - 0.5 kcal/mol or similar to 2-fold in chemistry at 37 degrees 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 k(2) 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 hig hly 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 nonhe lical RNA structure can be recognized by another RNA through tertiary interactions.