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 bon d 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 fun ctional groups involved in substrate recognition. By using a series of aden osine base analogues tagged with phosphorothioate substitutions, we identif y specific chemical groups involved in substrate binding. Only 10 adenosine s in the Escherichia coli ribozyme show significant sensitivity to interfer ence: A65, A66, A136, A232-234, A248, A249, A334, and A347. Most of these a denosine positions are universally conserved among all bacterial RNase P RN As; however, not all conserved adenosines are sensitive to analogue substit ution. Importantly, all but one of the sensitive nucleotides are located at positions of intermolecular cross-linking between the ribozyme and the sub strate. One site of interference that did not correlate with available stru ctural data involved A136 in J11/12. To confirm the generality of the resul ts, we repeated the interference analysis of J11/12 in the Bacillus subtili s RNase P ribozyme, which differs significantly in overall secondary struct ure. Notably, the B. subtilis ribozyme shows an identical interference patt ern at the position (A191) that is homologous to A136. Furthermore, mutatio n of A136 in the E. coli ribozyme gives rise to a measurable increase in th e equilibrium binding constant for the ribozyme-substrate interaction, whil e mutation of a nearby conserved nucleotide (A132) that is not sensitive to analogue incorporation does not. These results strongly support direct par ticipation of nucleotides in the P4, P11, J5/15, and J18/2 regions of riboz yme structure in pre-tRNA binding and implicate an additional region, J11/1 2, as involved in substrate recognition. In aggregate, the interference res ults provide a detailed chemical picture of how the conserved nucleotides a djacent to the pre-tRNA substrate contribute to substrate binding and provi de a framework for subsequent identification of the specific roles of these chemical groups in substrate recognition.