Identification of adenosine functional groups involved in substrate binding by the ribonuclease P ribozyme

The RNA component of bacterial ribonuclease P (RNase P) binds to substrate pre-tRNAs with high affinity and catalyzes site-specific phosphodiester bond hydrolysis to generate the mature tRNA 5' end. Herein we describe the use of biotinylated pre-tRNA substrates to isolate RNase P ribozyme-substrate complexes for nucleotide analogue interference mapping of ribozyme base functional groups involved in substrate recognition. By using a series of adenosine base analogues tagged with phosphorothioate substitutions, we identify specific chemical groups involved in substrate binding. Only 10 adenosines in the Escherichia coli ribozyme show significant sensitivity to interference: A65, A66, A136, A232-234, A248, A249, A334, and A347. Most of these adenosine positions are universally conserved among all bacterial RNase P RNAs; however, not all conserved adenosines are sensitive to analogue substitution. Importantly, all but one of the sensitive nucleotides are located at positions of intermolecular cross-linking between the ribozyme and the substrate. One site of interference that did not correlate with available structural data involved A136 in J11/12. To confirm the generality of the results, we repeated the interference analysis of J11/12 in the Bacillus subtilis RNase P ribozyme, which differs significantly in overall secondary structure. Notably, the B. subtilis ribozyme shows an identical interference pattern at the position (A191) that is homologous to A136. Furthermore, mutation of A136 in the E. coli ribozyme gives rise to a measurable increase in the equilibrium binding constant for the ribozyme-substrate interaction, while mutation of a nearby conserved nucleotide (A132) that is not sensitive to analogue incorporation does not. These results strongly support direct participation of nucleotides in the P4, P11, J5/15, and J18/2 regions of ribozyme structure in pre-tRNA binding and implicate an additional region, J11/12, as involved in substrate recognition. In aggregate, the interference results provide a detailed chemical picture of how the conserved nucleotides adjacent to the pre-tRNA substrate contribute to substrate binding and provide a framework for subsequent identification of the specific roles of these chemical groups in substrate recognition.