Mapping RNA-protein interactions in ribonuclease P from Escherichia coli using disulfide-linked EDTA-fe 1 1Edited by K. Nagai

The Bacillus subtilis RNase P holoenzyme contains two RNase P RNA and two RNase P protein subunits

“…The formation of the P RNA dimer in the holoenzyme is likely due to the presence of multiple RNA binding sites in the B. subtilis P protein+ Three binding sites have been inferred from its crystal structure (Stams et al+, 1998)+ Initially, one or more of these site in the P protein may bind one P RNA to form a specific (P RNA) 1 (P protein) 1 complex+ Subsequently, another site in the P protein in one (P RNA) 1 (P protein) 1 complex binds the P RNA in another (P RNA) 1 (P protein) 1 complex and vice versa+ This subsequent, symmetrical binding event results in a holoenzyme containing two P RNA and two P protein molecules (Fig+ 4, left)+ Crosslinking and affinity cleavage data suggest that two potential RNA-binding sites, the highly conserved "RNR" motif and the metal-binding loop, interact with P RNA in the (P RNA) 1 (P protein) 1 complex (Stams et al+, 1998;Biswas et al+, 2000; S+ Niranjanakumari & C+ A+ Fierke, unpubl+ results)+ Therefore, the best candidate for the RNA-binding site in the P protein responsible for P RNA dimerization is the same site involved in binding of the 59 leader of a pre-tRNA substrate (Niranjanakumari et al+, 1998b)+ This binding site in the P protein binds 4-5 single-stranded nucleotides with little sequence specificity+ In the absence of substrate, this site presumably binds a single-stranded region in the P RNA+ This proposed positioning of the P protein/P RNA contact in the dimer suggests that the affinity of substrates and products should be affected by dimerization+ Different oligomers form when the cognate B. subtilis P RNA is replaced with the noncognate, E. coli M1 RNA (Fig+ 4, right)+ The initial binding of the P protein to M1 RNA or to P RNA is probably similar, as suggested in numerous studies (Guerrier-Takada et al+, 1983;Reich et al+, 1988;Talbot & Altman 1994a)+ However, due to the variation in the shape of these RNAs, the subsequent binding event probably does not occur symmetrically for the noncognate E. coli M1 RNA-P protein complex+ Nonsymmetric interactions between (M1 RNA) 1 (P protein) 1 and (P RNA) 1 (P protein) 1 complexes would not enable both RNAs to interact with both proteins simultaneously+ Each P protein is free to interact with a third RNA, leading to aggregation, rather than to well-defined oligomeric species+…”

“…The dimerization of P RNA in the holoenzyme significantly affects the interpretation of previously published in vitro studies of the RNase P holoenzyme (Talbot & Altman, 1994a, 1994bLoria et al+, 1998;Gopalan et al+, 1999;Biswas et al+, 2000)+ Published protein-RNA affinity measurements are certainly affected by formation of a holoenzyme dimer+ Furthermore, dimerization could significantly influence the interpretation of experiments designed to identify P protein-binding sites+ For example, the P protein footprint on P RNA is much larger than expected for the small size of the P protein+ Our data suggests that some of the footprint may be derived from intersubunit RNA-RNA interactions formed upon dimerization+ Likewise, chemical crosslinking between the protein and the RNA should be reevaluated because the protein may crosslink to two different RNAs in the same complex+ Finally, mutational analysis of the P protein can be complicated due to the potential effects of the mutant on either P RNA binding or P RNA dimerization+…”

“…The holoenzyme of a bacterial RNase P is a ribonucleoprotein complex containing an RNA component (P RNA) of ;330-420 nt and a protein component (P protein) of ;120 amino acids (Frank & Pace, 1998;Altman & Kirsebom, 1999)+ The functional role of the protein component in the RNase P holoenzyme has been investigated extensively (Guerrier-Takada et al+, 1983, 1984Gardiner et al+, 1985;McClain et al+, 1987;Reich et al+, 1988;Peck-Miller & Altman, 1991;Svard & Kirsebom, 1992;Tallsjo & Kirsebom, 1993;Liu & Altman, 1994;Crary et al+, 1998;Kurz et al+, 1998;Loria et al+, 1998;Niranjanakumari et al+, 1998aNiranjanakumari et al+, , 1998bLoria & Pan, 1999)+ The Bacillus subtilis RNase P holoenzyme is remarkably efficient in the catalysis of precursor tRNA substrates with a k cat /K m near the diffusion limit (Kurz et al+, 1998;Reich et al+, 1988)+ In the absence of the protein component, k cat /K m decreases by 10 4 -fold under physiological conditions (Kurz et al+, 1998)+ A principal effect of the P protein function has been postulated to be the enhancement of substrate binding under physiological conditions (Crary et al+, 1998;Kurz et al+, 1998;Niranjanakumari et al+, 1998b)+ The physical state of the RNase P holoenzyme has received less attention+ Previous work by the Altman group conclusively showed that the Escherichia coli holoenzyme has an equal molar amount of RNA and protein (Talbot & Altman, 1994a)+ The affinity of the P protein binding to P RNA has been estimated to be ;0+5 nM, assuming a simple two-component binding isotherm (Talbot & Altman, 1994b)+ Functional studies by the Fierke group showed that the RNA-protein stoichiometry of the B. subtilis holoenzyme is also 1:1 (Niranjanakumari et al+, 1998a)+ Most studies in this area have focused on the details of P RNA-P protein interactions using chemical modification (Vioque et al+, 1988;Talbot & Altman, 1994b;Loria et al+, 1998;Biswas et al+, 20...…”

High-resolution structure of RNase P protein from Thermotoga maritima

“…The structural basis of any such interaction is not known. Moreover, there is no consistent structural model for the interaction between the protein and the RNase P RNA, despite several crosslink and footprint studies (11)(12)(13)(14).Phylogenetic comparative analysis has identified two major structural types of bacterial RNase P RNA (15). Both types have a homologous core structure that consists of about half the sequence lengths of the RNAs, but about half the sequence of each type of RNA has no homologous counterpart in the other RNA.…”

“…The structural basis of any such interaction is not known. Moreover, there is no consistent structural model for the interaction between the protein and the RNase P RNA, despite several crosslink and footprint studies (11)(12)(13)(14).…”