Modular architecture of eukaryotic RNase P and RNase MRP revealed by electron microscopy

Ribonuclease P (RNase P) and RNase MRP are closely related ribonucleoprotein enzymes, which process RNA substrates including tRNA precursors for RNase P and 5.8 S rRNA precursors, as well as some mRNAs, for RNase MRP. The structures of RNase P and RNase MRP have not yet been solved, so it is unclear how the proteins contribute to the structure of the complexes and how substrate specificity is determined. Using electron microscopy and image processing we show that eukaryotic RNase P and RNase MRP have a modular architecture, where proteins stabilize the RNA fold and contribute to cavities, channels and chambers between the modules. Such features are located at strategic positions for substrate recognition by shape and coordination of the cleaved-off sequence. These are also the sites of greatest difference between RNase P and RNase MRP, highlighting the importance of the adaptation of this region to the different substrates

Ribosomal protein L7Ae is a subunit of archaeal RNase P

To the mounting evidence of nonribosomal functions for ribosomal proteins, we now add L7Ae as a subunit of archaeal RNase P, a ribonucleoprotein (RNP) that catalyzes 5′-maturation of precursor tRNAs (pre-tRNAs). We first demonstrate that L7Ae coelutes with partially purified Methanococcus maripaludis (Mma) RNase P activity. After establishing in vitro reconstitution of the single RNA with four previously known protein subunits (POP5, RPP21, RPP29, and RPP30), we show that addition of L7Ae to this RNase P complex increases the optimal reaction temperature and kcat/Km (by ∼360-fold) for pre-tRNA cleavage to those observed with partially purified native Mma RNase P. We identify in the Mma RNase P RNA a putative kink-turn (K-turn), the structural motif recognized by L7Ae. The large stimulatory effect of Mma L7Ae on RNase P activity decreases to ≤ 4% of wild type upon mutating either the conserved nucleotides in this K-turn or amino acids in L7Ae shown to be essential for K-turn binding. The critical, multifunctional role of archaeal L7Ae in RNPs acting in tRNA processing (RNase P), RNA modification (H/ACA, C/D snoRNPs), and translation (ribosomes), especially by employing the same RNA-recognition surface, suggests coevolution of various translation-related functions, presumably to facilitate their coordinate regulation

Functional reconstitution and characterization of Pyrococcus furiosus RNase P

RNase P, which catalyzes the magnesium-dependent 5′-end maturation of tRNAs in all three domains of life, is composed of one essential RNA and a varying number of protein subunits depending on the source: at least one in bacteria, four in archaea, and nine in eukarya. To address why multiple protein subunits are needed for archaeal/eukaryal RNase P catalysis, in contrast to their bacterial relative, in vitro reconstitution of these holoenzymes is a prerequisite. Using recombinant subunits, we have reconstituted in vitro the RNase P holoenzyme from the thermophilic archaeon Pyroccocus furiosus (Pfu) and furthered our understanding regarding its functional organization and assembly pathway(s). Whereas Pfu RNase P RNA (RPR) alone is capable of multiple turnover, addition of all four RNase P protein (Rpp) subunits to Pfu RPR results in a 25-fold increase in its k(cat) and a 170-fold decrease in K(m). In fact, even in the presence of only one of two specific pairs of Rpps, the RPR displays activity at lower substrate and magnesium concentrations. Moreover, a pared-down, mini-Pfu RNase P was identified with an RPR deletion mutant. Results from our kinetic and footprinting studies on Pfu RNase P, together with insights from recent structures of bacterial RPRs, provide a framework for appreciating the role of multiple Rpps in archaeal RNase P

Structure of Pfu Pop5, an archaeal RNase P protein

We have used NMR spectroscopy and x-ray crystallography to determine the three-dimensional structure of PF1378 (Pfu Pop5), one of four protein subunits of archaeal RNase P that shares a homolog in the eukaryotic enzyme. RNase P is an essential and ubiquitous ribonucleoprotein enzyme required for maturation of tRNA. In bacteria, the enzyme's RNA subunit is responsible for cleaving the single-stranded 5′ leader sequence of precursor tRNA molecules (pre-tRNA), whereas the protein subunit assists in substrate binding. Although in bacteria the RNase P holoenzyme consists of one large catalytic RNA and one small protein subunit, in archaea and eukarya the enzyme contains several (≥4) protein subunits, each of which lacks sequence similarity to the bacterial protein. The functional role of the proteins is poorly understood, as is the increased complexity in comparison to the bacterial enzyme. Pfu Pop5 has been directly implicated in catalysis by the observation that it pairs with PF1914 (Pfu Rpp30) to functionally reconstitute the catalytic domain of the RNA subunit. The protein adopts an α–β sandwich fold highly homologous to the single-stranded RNA binding RRM domain. Furthermore, the three-dimensional arrangement of Pfu Pop5's structural elements is remarkably similar to that of the bacterial protein subunit. NMR spectra have been used to map the interaction of Pop5 with Pfu Rpp30. The data presented permit tantalizing hypotheses regarding the role of this protein subunit shared by archaeal and eukaryotic RNase P