Crystallization and preliminary X-ray diffraction analysis of the P3 RNA domain of yeast ribonuclease MRP in a complex with RNase P/MRP protein components Pop6 and Pop7

Abstract

Eukaryotic ribonucleases P and MRP are closely related RNA-based enzymes which contain a catalytic RNA component and several protein subunits. The roles of the protein subunits in the structure and function of eukaryotic ribonucleases P and MRP are not clear. Crystals of a complex that included a circularly permuted 46-nucleotide-long P3 domain of the RNA component of Saccharomyces cerevisiae ribonuclease MRP and selenomethionine derivatives of the shared ribonuclease P/MRP protein components Pop6 (18.2 kDa) and Pop7 (15.8 kDa) were obtained using the sitting-drop vapour-diffusion method. The crystals belonged to space group P4(2)22 (unit-cell parameters a = b = 127.2, c = 76.8 A, alpha = beta = gamma = 90 degrees ) and diffracted to 3.25 A resolution.

Figure 1

(a) The P3 domain of S. cerevisiae RNase MRP RNA; (b) a representative construct from the first set of P3-domain RNAs; (c) the construct from the second set of P3-domain RNAs which crystallized with the Pop6–Pop7 heterodimer.

Figure 2

Crystals of the P3-domain RNA complexed with Pop6–Pop7. (a) Before optimization of the crystallization conditions; (b) after optimization.

Figure 3

Analysis of the crystal content. (a) Protein analysis. Coomassie-stained 15% denaturing SDS–polyacrylamide gel. Lane 1, markers; lane 2, dissolved crystals; lane 3, purified Pop6–Pop7 heterodimer used in crystallization. Pop7 is known to have anomalously low mobility on SDS gels (Perederina et al., 2007 ▶). (b) RNA analysis. Ethidium bromide-stained 15% denaturing (8 M urea) polyacrylamide gel. Lane 1, markers; lane 2, dissolved crystals; lane 3, initial preparation of the P3-­domain RNA used in crystallization.

Figure 4

A representative image (1° oscillation) of the data collected from a crystal of the P3-domain RNA–Pop6–Pop7 complex. The image was collected using an ADSC Quantum 315 CCD detector on beamline X25 at NSLS, Brookhaven, USA.