Specific binding of a Pop6/Pop7 heterodimer to the P3 stem of the yeast RNase MRP and RNase P RNAs

Abstract

Pop6 and Pop7 are protein subunits of Saccharomyces cerevisiae RNase MRP and RNase P. Here we show that bacterially expressed Pop6 and Pop7 form a soluble heterodimer that binds the RNA components of both RNase MRP and RNase P. Footprint analysis of the interaction between the Pop6/7 heterodimer and the RNase MRP RNA, combined with gel mobility assays, demonstrates that the Pop6/7 complex binds to a conserved region of the P3 domain. Binding of these proteins to the MRP RNA leads to local rearrangement in the structure of the P3 loop and suggests that direct interaction of the Pop6/7 complex with the P3 domain of the RNA components of RNases MRP and P may mediate binding of other protein components. These results suggest a role for a key element in the RNase MRP and RNase P RNAs in protein binding, and demonstrate the feasibility of directly studying RNA-protein interactions in the eukaryotic RNases MRP and P complexes.

FIGURE 1.

Varying amounts (0.25–1 μg) of purified Pop6/7 complex fractionalized on a 15% SDS–polyacrylamide gel and stained with SYPRO Orange. Purified Pop6 was loaded as a control (left lane). Quantification of the intensities of the bands shows 1:1 Pop6: Pop7 molar ratio. Combined with Dynamic Light Scattering data (particle size 2.94 nm), this indicates formation of a heterodimer.

FIGURE 2.

Binding of Pop6/7 heterodimer to RNA. (A) Binding of Pop6/7 heterodimer to the full-length RNase MRP RNA (left lanes) or RNase P RNA (right lanes). Pop6/7 heterodimer binds both RNAs with 1:1 stoichiometry. (B) Binding of Pop6/7 heterodimer to the P3 domain from RNase MRP RNA (left lanes) or RNase P RNA (right lanes). Pop6/7 heterodimer binds both P3 domains. (C) Interaction of Pop6/7 with the mutated (U35A, U36A, A37U, C38G) P3 domain of RNase MRP. To determine the stoichiometry in A–C, the complexes were formed at concentrations of the components that were by at least an order of magnitude higher than the binding constant. The samples were loaded on 4% native polyacrylamide gels and fractionated at 4C; the gels were stained with toluidine blue. (D) Binding curves from filter-binding assays: binding with RNase MRP RNA (no competitor), triangles, red; with RNase MRP RNA (with 100X excess of tRNA), circles, blue; with P3 domain of RNase MRP (with 100X excess of tRNA), squares, black; with P3 domain of RNase P (with 100X excess of tRNA), squares, green.

FIGURE 3.

Footprint analysis of the Pop6/7–RNase MRP RNA complex, 6% denaturing (8 M urea) polyacrylamide gel. (Lanes 1,2) Control (untreated 5′-end labeled RNA) without and with Pop6/7; (lanes 3–8) digest with varying amounts of RNase V1 (lanes 3–5) without Pop6/7; (lanes 6–8) with Pop6/7; (lane 9) RNA digest with RNase T1 (G-ladder); (lane 10) alkali hydrolysis (ladder); (lanes 11–18) digest with varying amounts of RNase A (lanes 11–14) without Pop6/7; (lanes 15–18) with Pop6/7. The effects of binding of Pop6/7 heterodimer to RNase MRP RNA are confined to the P3 domain (marked on the right). Pop6/7 binding results in protection of nucleotides 30–38 (shown by a white box) and increases sensitivity of nucleotides G49, U76, and U77, to RNase V1 and U67 to RNase A.

FIGURE 4.

Expected secondary structures of the RNA components of S. cerevisiae RNase MRP (A) and RNase P (B). The diagrams and the nomenclature of the structural elements are based on Frank et al. (2000) and Li et al. (2002). (C) P3 domain of S. cerevisiae RNase MRP. The nucleotides common in RNase MRP and RNase P are circled. The nucleotides protected by Pop6/7 binding to RNase MRP RNA are marked by a bold line. The nucleotides that become more sensitive to nucleases upon Pop6/7 binding are marked by asterisks.