| Abstract
| - The RNA component of bacterial ribonuclease P (RNase P) binds to substrate pre-tRNAswith high affinity and catalyzes site-specific phosphodiester bond hydrolysis to generate the mature tRNA5‘ 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 groupsinvolved in substrate recognition. By using a series of adenosine base analogues tagged withphosphorothioate substitutions, we identify specific chemical groups involved in substrate binding. Only10 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 universallyconserved among all bacterial RNase P RNAs; however, not all conserved adenosines are sensitive toanalogue substitution. Importantly, all but one of the sensitive nucleotides are located at positions ofintermolecular cross-linking between the ribozyme and the substrate. One site of interference that did notcorrelate 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 differssignificantly in overall secondary structure. Notably, the B. subtilis ribozyme shows an identical interferencepattern at the position (A191) that is homologous to A136. Furthermore, mutation of A136 in the E. coliribozyme gives rise to a measurable increase in the equilibrium binding constant for the ribozyme-substrateinteraction, while mutation of a nearby conserved nucleotide (A132) that is not sensitive to analogueincorporation 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 chemicalpicture of how the conserved nucleotides adjacent to the pre-tRNA substrate contribute to substrate bindingand provide a framework for subsequent identification of the specific roles of these chemical groups insubstrate recognition.
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