Structure determination of RNase P and RNase MRP from Saccharomyces cerevisiae by electron mictroscopy and single particle image processing
Final Report Abstract
RNase P is a ribozyme that is conserved through all taxoniomic kingdoms of life. It processes t-RNA precursors. In bacteria the enzyme is comprised of a single RNA subunit, which is catalytically active and of a small protein subunit. In contrast to bacteria, eukaryotes have a far more complex enzyme with nine different protein subunits in addition to the RNA component. Furthermore, eukaryotes possess a second, closely related enzyme, which is called RNase MRP and processes mainly r-RNA precursors. RNase MRP has eight protein subunits in common with RNase P, and has a homologous RNA subunit and two specific protein subunits. We have used electron microscopy and single particle image processing to determine the structure of the eukaryotic RNase P and RNase MRP at 15-17Å resolution. The image reconstructions show that the two enzymes have a similar shape with a modular architecture that forms a large cavity in the center. This cavity is connected to a channel with various chambers. In RNase P the cavity matches t- RNA precursors in size and shape and would orient the t-RNA precursor in a way that its 3’ and 5’ ends point directly towards the entry of the channel. This suggests that the channel is probably involved in coordinating the leader sequences, which vary between different t-RNA precursors. In comparison, the cavity of RNase MRP is somewhat larger and its channel is replaced by an accessible slit. These differences are consistent with the different types of substrates which are processed by RNase P and RNase MRP. Fitting known high-resolution structures and models of sub-components of RNase P into the EM-maps shows where the subunits are positioned relative to the RNA subunit: The components which were acquired in eukaryotes are located at the extremities of the enzyme where they close the cavity and extend the length of the channel. This suggests that the main function of these gains is to enhance substrate recognition and binding. The subunits which were acquired in archaea group into two separate regions of the complex. One group binds tightly to the RNA subunit and probably stabilizes the structure of the RNA subunit in a more compact conformation than in the bacterial RNase P. The other group of subunits forms a module that provides the chambers and part of the channel Before this project was started a number of protein-protein interactions and information on the relative stoichiometry of the subunits in RNase P have been reported. Surprisingly the suggested stoichiometry could not be confirmed by our structural model. Furthermore, frequently reported protein-protein interactions appear unlikely in the light of the structural information reported here.
Publications
- Single-particle applications at intermediate resolution. Adv Protein Chem Struct Biol (2010) 81 61-88
B. Böttcher and K. Hipp
- Modular architecture of eukaryotic RNase P and RNase MRP revealed by electron microscopy. Nucleic Acids Res (2012) 40 3275-88
K. Hipp, K. Galani, C. Batisse, S. Prinz and B. Böttcher
(See online at https://doi.org/10.1093/nar/gkr1217)
