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1 Department of Chemistry and
2 Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, USA
Reprint requests to: Carol A. Fierke, Department of Chemistry, University of Michigan, 930 N. University, Ann Arbor, MI 48109, USA; e-mail: fierke{at}umich.edu; fax: (734) 647-4865.
Ribonuclease P (RNase P) is a ribonucleoprotein complex that catalyzes the cleavage of the 5' end of precursor tRNA. To characterize the interface between the Bacillus subtilis RNA (PRNA) and protein (P protein) components, the intraholoenzyme KD is determined as a function of ionic strength using a magnetocapture-based assay. Three distinct phases are evident. At low ionic strength, the affinity of PRNA for P protein is enhanced as the ionic strength increases mainly due to stabilization of the PRNA structure by cations. Lithium substitution in lieu of potassium enhances the affinity at low ionic strength, whereas the addition of ATP, known to stabilize the structure of P protein, does not affect the affinity. At high ionic strength, the observed affinity decreases as the ionic strength increases, consistent with disruption of ionic interactions. These data indicate that three to four ions are released on formation of holoenzyme, reflecting the number of ion pairs that occur between the P protein and PRNA. At moderate ionic strength, the two effects balance so that the apparent KD is not dependent on the ionic strength. The KD between the catalytic domain (C domain) and P protein has a similar triphasic dependence on ionic strength. Furthermore, the intraholoenzyme KD is identical to or tighter than that of full-length PRNA, demonstrating that the P protein binds solely to the C domain. Finally, pre-tRNAasp (but not tRNAasp) stabilizes the PRNA•P protein complex, as predicted by the direct interaction between the P protein and pre-tRNA leader.
Keywords: ribozyme; electrostatic; magnesium; cations; ribonuclease P
Abbreviations: C domain, catalytic domain of PRNA; C5, protein component of E. coli RNase P; E, RNase P holoenzyme; EDTA, (ethylenedinitrilo)-tetraacetic acid; M1 RNA, RNA component of E. coli RNase P; NTA, nitrilotriacetic acid; P, mature tRNA; P protein, protein component of B. subtilis RNase P; Pre-tRNA, precursor tRNA; PRNA, RNA component of B. subtilis RNase P; RNase P, ribonuclease P; RPR1 RNA, RNA component of S. cerevisiae RNase P; S or pre-tRNAAsp, precursor tRNAAsp; S domain, specificity domain of PRNA; Tris, Tris-(hydroxymethyl)-aminomethane
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