Andrey S. Krasilnikov

Crystal structure of the specificity domain of ribonuclease P

RNase P is the only endonuclease responsible for processing the 5′ end of transfer RNA by cleaving a precursor and leading to tRNA maturation. It contains an RNA component and a protein component and has been identified in all organisms. It was one of the first catalytic RNAs identified and the first that acts as a multiple-turnover enzyme in vivo. RNase P and the ribosome are so far the only two ribozymes known to be conserved in all kingdoms of life. The RNA component of bacterial RNase P can catalyse pre-tRNA cleavage in the absence of the RNase P protein in vitro and consists of two domains: a specificity domain and a catalytic domain. Here we report a 3.15-Å resolution crystal structure of the 154-nucleotide specificity domain of Bacillus subtilis RNase P. The structure reveals the architecture of this domain, the interactions that maintain the overall fold of the molecule, a large non-helical but well-structured module that is conserved in all RNase P RNA, and the regions that are involved in interactions with the substrate.

Crystal structure of the RNA component of bacterial ribonuclease P

Transfer RNA (tRNA) is produced as a precursor molecule that needs to be processed at its 3′ and 5′ ends. Ribonuclease P is the sole endonuclease responsible for processing the 5′ end of tRNA by cleaving the precursor and leading to tRNA maturation. It was one of the first catalytic RNA molecules identified and consists of a single RNA component in all organisms and only one protein component in bacteria. It is a true multi-turnover ribozyme and one of only two ribozymes (the other being the ribosome) that are conserved in all kingdoms of life. Here we show the crystal structure at 3.85 Å resolution of the RNA component of Thermotoga maritima ribonuclease P. The entire RNA catalytic component is revealed, as well as the arrangement of the two structural domains. The structure shows the general architecture of the RNA molecule, the inter- and intra-domain interactions, the location of the universally conserved regions, the regions involved in pre-tRNA recognition and the location of the active site. A model with bound tRNA is in agreement with all existing data and suggests the general basis for RNA–RNA recognition by this ribozyme.

Of proteins and RNA: The RNase P/MRP family

Nuclear ribonuclease (RNase) P is a ubiquitous essential ribonucleoprotein complex, one of only two known RNA-based enzymes found in all three domains of life. The RNA component is the catalytic moiety of RNases P across all phylogenetic domains; it contains a well-conserved core, whereas peripheral structural elements are diverse. RNA components of eukaryotic RNases P tend to be less complex than their bacterial counterparts, a simplification that is accompanied by a dramatic reduction of their catalytic ability in the absence of protein. The size and complexity of the protein moieties increase dramatically from bacterial to archaeal to eukaryotic enzymes, apparently reflecting the delegation of some structural functions from RNA to proteins and, perhaps, in response to the increased complexity of the cellular environment in the more evolutionarily advanced organisms; the reasons for the increased dependence on proteins are not clear. We review current information on RNase P and the closely related universal eukaryotic enzyme RNase MRP, focusing on their functions and structural organization.

Comparison of mitochondrial and nucleolar RNase MRP reveals identical RNA components with distinct enzymatic activities and protein components

RNase MRP is a ribonucleoprotein endoribonuclease found in three cellular locations where distinct substrates are processed: the mitochondria, the nucleolus, and the cytoplasm. Cytoplasmic RNase MRP is the nucleolar enzyme that is transiently relocalized during mitosis. Nucleolar RNase MRP (NuMRP) was purified to homogeneity, and we extensively purified the mitochondrial RNase MRP (MtMRP) to a single RNA component identical to the NuMRP RNA. Although the protein components of the NuMRP were identified by mass spectrometry successfully, none of the known NuMRP proteins were found in the MtMRP preparation. Only trace amounts of the core NuMRP protein, Pop4, were detected in MtMRP by Western blot. In vitro activity of the two enzymes was compared. MtMRP cleaved only mitochondrial ORI5 substrate, while NuMRP cleaved all three substrates. However, the NuMRP enzyme cleaved the ORI5 substrate at sites different than the MtMRP enzyme. In addition, enzymatic differences in preferred ionic strength confirm these enzymes as distinct entities. Magnesium was found to be essential to both enzymes. We tested a number of reported inhibitors including puromycin, pentamidine, lithium, and pAp. Puromycin inhibition suggested that it binds directly to the MRP RNA, reaffirming the role of the RNA component in catalysis. In conclusion, our study confirms that the NuMRP and MtMRP enzymes are distinct entities with differing activities and protein components but a common RNA subunit, suggesting that the RNA must be playing a crucial role in catalytic activity.

Footprinting analysis demonstrates extensive similarity between eukaryotic RNase P and RNase MRP holoenzymes

Eukaryotic ribonuclease (RNase) P and RNase MRP are evolutionary related RNA-based enzymes involved in metabolism of various RNA molecules, including tRNA and rRNA. In contrast to the closely related eubacterial RNase P, which is comprised of an RNA component and a single small protein, these enzymes contain multiple protein components. Here we report the results of footprinting studies performed on purified Saccharomyces cerevisiae RNase MRP and RNase P holoenzymes. The results identify regions of the RNA components affected by the protein moiety, suggest a role of the proteins in stabilization of the RNA fold, and point to substantial similarities between the two evolutionary related RNA-based enzymes.

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

Eukaryotic ribonuclease (RNase) P and RNase MRP are closely related ribonucleoprotein complexes involved in the metabolism of various RNA molecules including tRNA, rRNA, and some mRNAs. While evolutionarily related to bacterial RNase P, eukaryotic enzymes of the RNase P/MRP family are much more complex. Saccharomyces cerevisiae RNase P consists of a catalytic RNA component and nine essential proteins; yeast RNase MRP has an RNA component resembling that in RNase P and 10 essential proteins, most of which are shared with RNase P. The structural organizations of eukaryotic RNases P/MRP are not clear. Here we present the results of RNA-protein UV crosslinking studies performed on RNase P and RNase MRP holoenzymes isolated from yeast. The results indicate locations of specific protein-binding sites in the RNA components of RNase P and RNase MRP and shed light on the structural organizations of these large ribonucleoprotein complexes.

The P3 domain of eukaryotic RNases P/MRP: making a protein-rich RNA-based enzyme.

Nuclear Ribonuclease (RNase) P is a universal essential RNA-based enzyme made of a catalytic RNA component and a protein part; eukaryotic RNase P is closely related to a universal eukaryotic ribonucleoprotein RNase MRP. The protein part of the eukaryotic RNases P/MRP is dramatically more complex than that in bacterial and archaeal RNases P. The increase in the complexity of the protein part in eukaryotic RNases P/MRP was accompanied by the appearance of a novel structural element in the RNA component: an essential and phylogenetically conserved helix-loop-helix P3 RNA domain. The crystal structure of the P3 RNA domain in a complex with protein components Pop6 and Pop7 has been recently solved. Here we discuss the most salient structural features of the P3 domain as well as its possible role in the evolutionary transition to the protein-rich eukaryotic RNases P/MRP.

Footprinting analysis of interactions between the largest eukaryotic RNase P/MRP protein Pop1 and RNase P/MRP RNA components

Ribonuclease (RNase) P and RNase MRP are closely related catalytic ribonucleoproteins involved in the metabolism of a wide range of RNA molecules, including tRNA, rRNA, and some mRNAs. The catalytic RNA component of eukaryotic RNase P retains the core elements of the bacterial RNase P ribozyme; however, the peripheral RNA elements responsible for the stabilization of the global architecture are largely absent in the eukaryotic enzyme. At the same time, the protein makeup of eukaryotic RNase P is considerably more complex than that of the bacterial RNase P. RNase MRP, an essential and ubiquitous eukaryotic enzyme, has a structural organization resembling that of eukaryotic RNase P, and the two enzymes share most of their protein components. Here, we present the results of the analysis of interactions between the largest protein component of yeast RNases P/MRP, Pop1, and the RNA moieties of the enzymes, discuss structural implications of the results, and suggest that Pop1 plays the role of a scaffold for the stabilization of the global architecture of eukaryotic RNase P RNA, substituting for the network of RNA-RNA tertiary interactions that maintain the global RNA structure in bacterial RNase P.

Interactions of a Pop5/Rpp1 heterodimer with the catalytic domain of RNase MRP.

Ribonuclease (RNase) MRP is a multicomponent ribonucleoprotein complex closely related to RNase P. RNase MRP and eukaryotic RNase P share most of their protein components, as well as multiple features of their catalytic RNA moieties, but have distinct substrate specificities. While RNase P is practically universally found in all three domains of life, RNase MRP is essential in eukaryotes. The structural organizations of eukaryotic RNase P and RNase MRP are poorly understood. Here, we show that Pop5 and Rpp1, protein components found in both RNase P and RNase MRP, form a heterodimer that binds directly to the conserved area of the putative catalytic domain of RNase MRP RNA. The Pop5/Rpp1 binding site corresponds to the protein binding site in bacterial RNase P RNA. Structural and evolutionary roles of the Pop5/Rpp1 heterodimer in RNases P and MRP are discussed.

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

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.