Cleavage of model substrates by archaeal RNase P: role of protein cofactors in cleavage-site selection

Abstract

RNase P is a catalytic ribonucleoprotein primarily involved in tRNA biogenesis. Archaeal RNase P comprises a catalytic RNase P RNA (RPR) and at least four protein cofactors (RPPs), which function as two binary complexes (POP5•RPP30 and RPP21• RPP29). Exploiting the ability to assemble a functional Pyrococcus furiosus (Pfu) RNase P in vitro, we examined the role of RPPs in influencing substrate recognition by the RPR. We first demonstrate that Pfu RPR, like its bacterial and eukaryal counterparts, cleaves model hairpin loop substrates albeit at rates 90- to 200-fold lower when compared with cleavage by bacterial RPR, highlighting the functionally comparable catalytic cores in bacterial and archaeal RPRs. By investigating cleavage-site selection exhibited by Pfu RPR (±RPPs) with various model substrates missing consensus-recognition elements, we determined substrate features whose recognition is facilitated by either POP5•RPP30 or RPP21•RPP29 (directly or indirectly via the RPR). Our results also revealed that Pfu RPR + RPP21•RPP29 displays substrate-recognition properties coinciding with those of the bacterial RPR-alone reaction rather than the Pfu RPR, and that this behaviour is attributable to structural differences in the substrate-specificity domains of bacterial and archaeal RPRs. Moreover, our data reveal a hierarchy in recognition elements that dictates cleavage-site selection by archaeal RNase P.

Structural organizations of yeast RNase P and RNase MRP holoenzymes as revealed by UV-crosslinking studies of RNA–protein interactions

“…3). It was previously suggested ) that in archaeal RNase P, the protein component Rpp29 (a homolog of yeast Pop4) acting in a pair with the protein component Rpp21 (a homolog of yeast Rpr2) (Tsai et al 2006;Xu et al 2009;Sinapah et al 2011) serves to enable long-distance interactions between the catalytic (C-) and the specificity (S-) domains of the RNA component of RNase P. Mapping of the crosslinking sites of Pop4 into the expected outline of the RNA component of yeast RNase P (Fig. 3) demonstrates that the localization of Pop4 is consistent with the protein's role in the stabilization of the mutual orientation of the C-domain and S-domain of yeast RNase P RNA.…”

Functional Coupling between a Distal Interaction and the Cleavage Site in Bacterial RNase-P-RNA-Mediated Cleavage

“…It is therefore noteworthy to mention that the Hill coefficient in the cleavage of pATSerUG by P. furiosus RPR was recently determined to be ≈ 4. 10 Interaction between the 3′ end of the substrate and RPR suppresses cleavage at −1 Residue G + 73 in the substrate interacts with U 294 in the RPR-substrate complex (referred to as the + 73/294 interaction; 29 see also Reiter et al 18 ). Replacing U at position 294 with C seems to facilitate the disruption of the C − 1 /G + 73 pair.…”

“…S1). 10,31,32 Moreover, ES 1 to ES 1⁎ describes, for example, the positioning of Mg 2+ that results in cleavage at − 1, while ES 1 to ES 2 accounts for, for example, the opening of C − 1 /G 73 (when present) and the positioning of Mg 2+ resulting in cleavage at +1. Note also that there is no precedent for the reverse reaction [i.e., formation of ES 2 from EP (or from EP ⁎ to ES 1⁎ )], hence k − 3 (and k − 3 ⁎ ) and the arrows are placed in parentheses.…”

“…[1][2][3][4][5] Regardless of origin, RPR alone, without protein, is capable of cleaving various substrates. [6][7][8][9][10] Recent reports, however, demonstrate protein-based RNase-P-like activity. 11,12 On the basis of its secondary structure, RPR is divided into two major domains: specificity domain (S-domain) and catalytic domain (C-domain).…”

“…1; see also the text below). 10,22 On the basis of these data, we provided a model in which a productive TSL/TBS interaction influences the positioning of chemical groups and/or Mg 2+ at the cleavage site. 22 Given that the GAAA tetraloop adopts a characteristic structural topography, [23][24][25][26] we decided to change its structure and to study the effect on cleavage.…”