External Guide Sequence Effectively Suppresses the Gene Expression and Replication of Herpes Simplex Virus 2.

Ribonuclease P (RNase P) complexed with an external guide sequence (EGS) represents a promising nucleic acid-based gene targeting approach for gene expression knock-down and modulation. The RNase P-EGS strategy is unique as an EGS can be designed to basepair any mRNA sequence and recruit intracellular RNase P for hydrolysis of the target mRNA. In this study, we provide the first direct evidence that the RNase P-based approach effectively blocks the gene expression and replication of herpes simplex virus 2 (HSV-2), the causative agent of genital herpes. We constructed EGSs to target the mRNA encoding HSV-2 single-stranded DNA binding protein ICP8, which is essential for viral DNA genome replication and growth. In HSV-2 infected cells expressing a functional EGS, ICP8 levels were reduced by 85%, and viral growth decreased by 3000 folds. On the contrary, ICP8 expression and viral growth exhibited no substantial differences between cells expressing no EGS and those expressing a disabled EGS with mutations precluding RNase P recognition. The anti-ICP8 EGS is specific in targeting ICP8 because it only affects ICP8 expression but does not affect the expression of the other viral immediate-early and early genes examined. This study shows the effective and specific anti-HSV-2 activity of the RNase P-EGS approach and demonstrates the potential of EGS RNAs for anti-HSV-2 applications.

Multiple structural flavors of RNase P in precursor tRNA processing.

The precursor transfer RNAs (pre-tRNAs) require extensive processing to generate mature tRNAs possessing proper fold, structural stability, and functionality required to sustain cellular viability. The road to tRNA maturation follows an ordered process: 5'-processing, 3'-processing, modifications at specific sites, if any, and 3'-CCA addition before aminoacylation and recruitment to the cellular protein synthesis machinery. Ribonuclease P (RNase P) is a universally conserved endonuclease in all domains of life, performing the hydrolysis of pre-tRNA sequences at the 5' end by the removal of phosphodiester linkages between nucleotides at position -1 and +1. Except for an archaeal species: Nanoarchaeum equitans where tRNAs are transcribed from leaderless-position +1, RNase P is indispensable for life and displays fundamental variations in terms of enzyme subunit composition, mechanism of substrate recognition and active site architecture, utilizing in all cases a two metal ion-mediated conserved catalytic reaction. While the canonical RNA-based ribonucleoprotein RNase P has been well-known to occur in bacteria, archaea, and eukaryotes, the occurrence of RNA-free protein-only RNase P in eukaryotes and RNA-free homologs of Aquifex RNase P in prokaryotes has been discovered more recently. This review aims to provide a comprehensive overview of structural diversity displayed by various RNA-based and RNA-free RNase P holoenzymes towards harnessing critical RNA-protein and protein-protein interactions in achieving conserved pre-tRNA processing functionality. Furthermore, alternate roles and functional interchangeability of RNase P are discussed in the context of its employability in several clinical and biotechnological applications. This article is categorized under: RNA Processing > tRNA Processing RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

The protein-only RNase Ps, endonucleases that cleave pre-tRNA: Biological relevance, molecular architectures, substrate recognition and specificity, and protein interactomes.

Protein-only RNase P (PRORP) is an essential enzyme responsible for the 5' maturation of precursor tRNAs (pre-tRNAs). PRORPs are classified into three categories with unique molecular architectures, although all three classes of PRORPs share a mechanism and have similar active sites. Single subunit PRORPs, like those found in plants, have multiple isoforms with different localizations, substrate specificities, and temperature sensitivities. Most recently, Arabidopsis thaliana PRORP2 was shown to interact with TRM1A and B, highlighting a new potential role between these enzymes. Work with At PRORPs led to the development of a ribonuclease that is being used to protect against plant viruses. The mitochondrial RNase P complex, found in metazoans, consists of PRORP, TRMT10C, and SDR5C1, and has also been shown to have substrate specificity, although the cause is unknown. Mutations in mitochondrial tRNA and mitochondrial RNase P have been linked to human disease, highlighting the need to continue understanding this complex. The last class of PRORPs, homologs of Aquifex RNase P (HARPs), is found in thermophilic archaea and bacteria. This most recently discovered type of PRORP forms a large homo-oligomer complex. Although numerous structures of HARPs have been published, it is still unclear how HARPs bind pre-tRNAs and in what ratio. There is also little investigation into the substrate specificity and ideal conditions for HARPs. Moving forward, further work is required to fully characterize each of the three classes of PRORP, the pre-tRNA binding recognition mechanism, the rules of substrate specificity, and how these three distinct classes of PRORP evolved. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems.

Discovery, structure, mechanisms, and evolution of protein-only RNase P enzymes.

The endoribonuclease RNase P is responsible for tRNA 5' maturation in all domains of life. A unique feature of RNase P is the variety of enzyme architectures, ranging from dual- to multi-subunit ribonucleoprotein forms with catalytic RNA subunits to protein-only enzymes, the latter occurring as single- or multi-subunit forms or homo-oligomeric assemblies. The protein-only enzymes evolved twice: a eukaryal protein-only RNase P termed PRORP and a bacterial/archaeal variant termed homolog of Aquifex RNase P (HARP); the latter replaced the RNA-based enzyme in a small group of thermophilic bacteria but otherwise coexists with the ribonucleoprotein enzyme in a few other bacteria as well as in those archaea that also encode a HARP. Here we summarize the history of the discovery of protein-only RNase P enzymes and review the state of knowledge on structure and function of bacterial HARPs and eukaryal PRORPs, including human mitochondrial RNase P as a paradigm of multi-subunit PRORPs. We also describe the phylogenetic distribution and evolution of PRORPs, as well as possible reasons for the spread of PRORPs in the eukaryal tree and for the recruitment of two additional protein subunits to metazoan mitochondrial PRORP. We outline potential applications of PRORPs in plant biotechnology and address diseases associated with mutations in human mitochondrial RNase P genes. Finally, we consider possible causes underlying the displacement of the ancient RNA enzyme by a protein-only enzyme in a small group of bacteria.

Coevolution of RNA and protein subunits in RNase P and RNase MRP, two RNA processing enzymes.

RNase P and RNase mitochondrial RNA processing (MRP) are ribonucleoproteins (RNPs) that consist of a catalytic RNA and a varying number of protein cofactors. RNase P is responsible for precursor tRNA maturation in all three domains of life, while RNase MRP, exclusive to eukaryotes, primarily functions in rRNA biogenesis. While eukaryotic RNase P is associated with more protein cofactors and has an RNA subunit with fewer auxiliary structural elements compared to its bacterial cousin, the double-anchor precursor tRNA recognition mechanism has remarkably been preserved during evolution. RNase MRP shares evolutionary and structural similarities with RNase P, preserving the catalytic core within the RNA moiety inherited from their common ancestor. By incorporating new protein cofactors and RNA elements, RNase MRP has established itself as a distinct RNP capable of processing ssRNA substrates. The structural information on RNase P and MRP helps build an evolutionary trajectory, depicting how emerging protein cofactors harmonize with the evolution of RNA to shape different functions for RNase P and MRP. Here, we outline the structural and functional relationship between RNase P and MRP to illustrate the coevolution of RNA and protein cofactors, a key driver for the extant, diverse RNP world.

The previously uncharacterized RnpM (YlxR) protein modulates the activity of ribonuclease P in Bacillus subtilis in vitro.

Even though Bacillus subtilis is one of the most studied organisms, no function has been identified for about 20% of its proteins. Among these unknown proteins are several RNA- and ribosome-binding proteins suggesting that they exert functions in cellular information processing. In this work, we have investigated the RNA-binding protein YlxR. This protein is widely conserved in bacteria and strongly constitutively expressed in B. subtilis suggesting an important function. We have identified the RNA subunit of the essential RNase P as the binding partner of YlxR. The main activity of RNase P is the processing of 5' ends of pre-tRNAs. In vitro processing assays demonstrated that the presence of YlxR results in reduced RNase P activity. Chemical cross-linking studies followed by in silico docking analysis and experiments with site-directed mutant proteins suggest that YlxR binds to the region of the RNase P RNA that is important for binding and cleavage of the pre-tRNA substrate. We conclude that the YlxR protein is a novel interaction partner of the RNA subunit of RNase P that serves to finetune RNase P activity to ensure appropriate amounts of mature tRNAs for translation. We rename the YlxR protein RnpM for RNase P modulator.

The specificity landscape of bacterial ribonuclease P.

Developing quantitative models of substrate specificity for RNA processing enzymes is a key step toward understanding their biology and guiding applications in biotechnology and biomedicine. Optimally, models to predict relative rate constants for alternative substrates should integrate an understanding of structures of the enzyme bound to "fast" and "slow" substrates, large datasets of rate constants for alternative substrates, and transcriptomic data identifying in vivo processing sites. Such data are either available or emerging for bacterial ribonucleoprotein RNase P a widespread and essential tRNA 5' processing endonuclease, thus making it a valuable model system for investigating principles of biological specificity. Indeed, the well-established structure and kinetics of bacterial RNase P enabled the development of high throughput measurements of rate constants for tRNA variants and provided the necessary framework for quantitative specificity modeling. Several studies document the importance of conformational changes in the precursor tRNA substrate as well as the RNA and protein subunits of bacterial RNase P during binding, although the functional roles and dynamics are still being resolved. Recently, results from cryo-EM studies of E. coli RNase P with alternative precursor tRNAs are revealing prospective mechanistic relationships between conformational changes and substrate specificity. Yet, extensive uncharted territory remains, including leveraging these advances for drug discovery, achieving a complete accounting of RNase P substrates, and understanding how the cellular context contributes to RNA processing specificity in vivo.

A tRNA-modifying enzyme facilitates RNase P activity in Arabidopsis nuclei.

RNase P is the essential activity that performs the 5' maturation of transfer RNA (tRNA) precursors. Beyond the ancestral form of RNase P containing a ribozyme, protein-only RNase P enzymes termed PRORP were identified in eukaryotes. In human mitochondria, PRORP forms a complex with two protein partners to become functional. In plants, although PRORP enzymes are active alone, we investigate their interaction network to identify potential tRNA maturation complexes. Here we investigate functional interactions involving the Arabidopsis nuclear RNase P PRORP2. We show, using an immuno-affinity strategy, that PRORP2 occurs in a complex with the tRNA methyl transferases TRM1A and TRM1B in vivo. Beyond RNase P, these enzymes can also interact with RNase Z. We show that TRM1A/TRM1B localize in the nucleus and find that their double knockout mutation results in a severe macroscopic phenotype. Using a combination of immuno-detections, mass spectrometry and a transcriptome-wide tRNA sequencing approach, we observe that TRM1A/TRM1B are responsible for the m22G26 modification of 70% of cytosolic tRNAs in vivo. We use the transcriptome wide tRNAseq approach as well as RNA blot hybridizations to show that RNase P activity is impaired in TRM1A/TRM1B mutants for specific tRNAs, in particular, tRNAs containing a m22G modification at position 26 that are strongly downregulated in TRM1A/TRM1B mutants. Altogether, results indicate that the m22G-adding enzymes TRM1A/TRM1B functionally cooperate with nuclear RNase P in vivo for the early steps of cytosolic tRNA biogenesis.

Raising NAD+ Level Stimulates Short-Chain Dehydrogenase/Reductase Proteins to Alleviate Heart Failure Independent of Mitochondrial Protein Deacetylation.

BACKGROUND: Strategies to increase cellular NAD+ (oxidized nicotinamide adenine dinucleotide) level have prevented cardiac dysfunction in multiple models of heart failure, but molecular mechanisms remain unclear. Little is known about the benefits of NAD+-based therapies in failing hearts after the symptoms of heart failure have appeared. Most pretreatment regimens suggested mechanisms involving activation of sirtuin, especially Sirt3 (sirtuin 3), and mitochondrial protein acetylation. METHODS: We induced cardiac dysfunction by pressure overload in SIRT3-deficient (knockout) mice and compared their response with nicotinamide riboside chloride treatment with wild-type mice. To model a therapeutic approach, we initiated the treatment in mice with established cardiac dysfunction. RESULTS: We found nicotinamide riboside chloride improved mitochondrial function and blunted heart failure progression. Similar benefits were observed in wild-type and knockout mice. Boosting NAD+ level improved the function of NAD(H) redox-sensitive SDR (short-chain dehydrogenase/reductase) family proteins. Upregulation of Mrpp2 (mitochondrial ribonuclease P protein 2), a multifunctional SDR protein and a subunit of mitochondrial ribonuclease P, improves mitochondrial DNA transcripts processing and electron transport chain function. Activation of SDRs in the retinol metabolism pathway stimulates RXRα (retinoid X receptor α)/PPARα (proliferator-activated receptor α) signaling and restores mitochondrial oxidative metabolism. Downregulation of Mrpp2 and impaired mitochondrial ribonuclease P were found in human failing hearts, suggesting a shared mechanism of defective mitochondrial biogenesis in mouse and human heart failure. CONCLUSIONS: These findings identify SDR proteins as important regulators of mitochondrial function and molecular targets of NAD+-based therapy. Furthermore, the benefit is observed regardless of Sirt3-mediated mitochondrial protein deacetylation, a widely held mechanism for NAD+-based therapy for heart failure. The data also show that NAD+-based therapy can be useful in pre-existing heart failure.

Bacterial RNA-free RNase P: Structural and functional characterization of multiple oligomeric forms of a minimal protein-only ribonuclease P.

tRNAs are typically transcribed with extended 5' and 3' ends that must be removed before they attain their active form. One of the first steps of tRNA processing in nearly every organism is the removal of the 5' leader sequence by ribonuclease P (RNase P). Here, we investigate a recently discovered class of RNase P enzymes, Homologs of Aquifex RNase P (HARPs). In contrast to other RNase Ps, HARPs consist only of a metallonuclease domain and lack the canonical substrate recognition domain essential in other classes of proteinaceous RNase P. We determined the cryo-EM structure of Aquifex aeolicus HARP (Aq880) and two crystal structures of Hydrogenobacter thermophilus HARP (Hth1307) to reveal that both enzymes form large ring-like assemblies: a dodecamer in Aq880 and a tetradecamer in Hth1307. In both oligomers, the enzyme active site is 42 Å away from a positively charged helical region, as seen in other protein-only RNase P enzymes, which likely serves to recognize and bind the elbow region of the pre-tRNA substrate. In addition, we use native mass spectrometry to confirm and characterize the previously unreported tetradecamer state. Notably, we find that multiple oligomeric states of Hth1307 are able to cleave pre-tRNAs. Furthermore, our single-turnover kinetic studies indicate that Hth1307 cleaves pre-tRNAs from multiple species with a preference for native substrates. These data provide a closer look at the nuanced similarities and differences in tRNA processing across disparate classes of RNase P.

Cleavage kinetics of human mitochondrial RNase P and contribution of its non-nuclease subunits.

RNase P is the endonuclease responsible for the 5' processing of precursor tRNAs (pre-tRNAs). Unlike the single-subunit protein-only RNase P (PRORP) found in plants or protists, human mitochondrial RNase P is a multi-enzyme assembly that in addition to the homologous PRORP subunit comprises a methyltransferase (TRMT10C) and a dehydrogenase (SDR5C1) subunit; these proteins, but not their enzymatic activities, are required for efficient pre-tRNA cleavage. Here we report a kinetic analysis of the cleavage reaction by human PRORP and its interplay with TRMT10C-SDR5C1 including 12 different mitochondrial pre-tRNAs. Surprisingly, we found that PRORP alone binds pre-tRNAs with nanomolar affinity and can even cleave some of them at reduced efficiency without the other subunits. Thus, the ancient binding mode, involving the tRNA elbow and PRORP's PPR domain, appears basically retained by human PRORP, and its metallonuclease domain is in principle correctly folded and functional. Our findings support a model according to which the main function of TRMT10C-SDR5C1 is to direct PRORP's nuclease domain to the cleavage site, thereby increasing the rate and accuracy of cleavage. This functional dependence of human PRORP on an extra tRNA-binding protein complex likely reflects an evolutionary adaptation to the erosion of canonical structural features in mitochondrial tRNAs.

Human RNase P exhibits and controls distinct ribonucleolytic activities required for ordered maturation of tRNA.

Precursor tRNAs are transcribed with flanking and intervening sequences known to be processed by specific ribonucleases. Here, we show that transcription complexes of RNA polymerase III assembled on tRNA genes comprise RNase P that cleaves precursor tRNA and subsequently degrades the excised 5' leader. Degradation is based on a 3'-5' exoribonucleolytic activity carried out by the protein subunit Rpp14, as determined by biochemical and reverse genetic analyses. Neither reconstituted nor purified RNase P displays this magnesium ion-dependent, processive exoribonucleolytic activity. Markedly, knockdown of Rpp14 by RNA interference leads to a wide-ranging inhibition of cleavage of flanking and intervening sequences of various precursor tRNAs in extracts and cells. This study reveals that RNase P controls tRNA splicing complex and RNase Z for ordered maturation of nascent precursor tRNAs by transcription complexes.

Mapping the MOB proteins' proximity network reveals a unique interaction between human MOB3C and the RNase P complex.

Distinct functions mediated by members of the monopolar spindle-one-binder (MOB) family of proteins remain elusive beyond the evolutionarily conserved and well-established roles of MOB1 (MOB1A/B) in regulating tissue homeostasis within the Hippo pathway. Since MOB proteins are adaptors, understanding how they engage in protein-protein interactions and help assemble complexes is essential to define the full scope of their biological functions. To address this, we undertook a proximity-dependent biotin identification approach to define the interactomes of all seven human MOB proteins in HeLa and human embryonic kidney 293 cell lines. We uncovered >200 interactions, of which at least 70% are unreported on BioGrid. The generated dataset reliably recalled the bona fide interactors of the well-studied MOBs. We further defined the common and differential interactome between different MOBs on a subfamily and an individual level. We discovered a unique association between MOB3C and 7 of 10 protein subunits of the RNase P complex, an endonuclease that catalyzes tRNA 5' maturation. As a proof of principle for the robustness of the generated dataset, we validated the specific interaction of MOB3C with catalytically active RNase P by using affinity purification-mass spectrometry and pre-tRNA cleavage assays of MOB3C pulldowns. In summary, our data provide novel insights into the biology of MOB proteins and reveal the first interactors of MOB3C, components of the RNase P complex, and hence an exciting nexus with RNA biology.

Mapping of RNase P Ribozyme Regions in Proximity with a Human RNase P Subunit Protein Using Fe(II)-EDTA Cleavage and Nuclease Footprint Analyses.

Ribonuclease P (RNase P), which may consist of both protein subunits and a catalytic RNA part, is responsible for 5' maturation of tRNA by cleaving the 5'-leader sequence. In Escherichia coli, RNase P contains a catalytic RNA subunit (M1 RNA) and a protein factor (C5 protein). In human cells, RNase P holoenzyme consists of an RNA subunit (H1 RNA) and multiple protein subunits that include human RPP29 protein. M1GS, a sequence specific targeting ribozyme derived from M1 RNA, can be constructed to target a specific mRNA to degrade it in vitro. Recent studies have shown that M1GS ribozymes are efficient in blocking the expression of viral mRNAs in cultured cells and in animals. These results suggest that RNase P ribozymes have the potential to be useful in basic research and in clinical applications. It has been shown that RNase P binding proteins, such as C5 protein and RPP29, can enhance the activities of M1GS RNA in processing a natural tRNA substrate and a target mRNA. Understanding how RPP29 binds to M1GS RNA and enhances the enzyme's catalytic activity will provide great insight into developing more robust gene-targeting ribozymes for in vivo application. In this chapter, we describe the methods of using Fe(II)-ethylenediaminetetraacetic acid (EDTA) cleavage and nuclease footprint analyses to determine the regions of a M1GS ribozyme that are in proximity to RPP29 protein.

Suppressing Kaposi's Sarcoma-Associated Herpesvirus Lytic Gene Expression and Replication by RNase P Ribozyme.

Kaposi's sarcoma, an AIDS-defining illness, is caused by Kaposi's sarcoma-associated herpesvirus (KSHV), an oncogenic virus. In this study, we engineered ribozymes derived from ribonuclease P (RNase P) catalytic RNA with targeting against the mRNA encoding KSHV immediate early replication and transcription activator (RTA), which is vital for KSHV gene expression. The functional ribozyme F-RTA efficiently sliced the RTA mRNA sequence in vitro. In cells, KSHV production was suppressed with ribozyme F-RTA expression by 250-fold, and RTA expression was suppressed by 92-94%. In contrast, expression of control ribozymes hardly affected RTA expression or viral production. Further studies revealed both overall KSHV early and late gene expression and viral growth decreased because of F-RTA-facilitated suppression of RTA expression. Our results indicate the first instance of RNase P ribozymes having potential for use in anti-KSHV therapy.

Characterization of RNA-based and protein-only RNases P from bacteria encoding both enzyme types.

A small group of bacteria encode two types of RNase P, the classical ribonucleoprotein (RNP) RNase P as well as the protein-only RNase P HARP (homolog of Aquifex RNase P). We characterized the dual RNase P activities of five bacteria that belong to three different phyla. All five bacterial species encode functional RNA (gene rnpB) and protein (gene rnpA) subunits of RNP RNase P, but only the HARP of the thermophile Thermodesulfatator indicus (phylum Thermodesulfobacteria) was found to have robust tRNA 5'-end maturation activity in vitro and in vivo in an Escherichia coli RNase P depletion strain. These findings suggest that both types of RNase P are able to contribute to the essential tRNA 5'-end maturation activity in T. indicus, thus resembling the predicted evolutionary transition state in the progenitor of the Aquificaceae before the loss of rnpA and rnpB genes in this family of bacteria. Remarkably, T. indicus RNase P RNA is transcribed with a P12 expansion segment that is posttranscriptionally excised in vivo, such that the major fraction of the RNA is fragmented and thereby truncated by ∼70 nt in the native T. indicus host as well as in the E. coli complementation strain. Replacing the native P12 element of T. indicus RNase P RNA with the short P12 helix of Thermotoga maritima RNase P RNA abolished fragmentation, but simultaneously impaired complementation efficiency in E. coli cells, suggesting that intracellular fragmentation and truncation of T. indicus RNase P RNA may be beneficial to RNA folding and/or enzymatic activity.

Human RNase P: overview of a ribonuclease of interrelated molecular networks and gene-targeting systems.

The seminal discovery of ribonuclease P (RNase P) and its catalytic RNA by Sidney Altman has not only revolutionized our understanding of life, but also opened new fields for scientific exploration and investigation. This review focuses on human RNase P and its use as a gene-targeting tool, two topics initiated in Altman's laboratory. We outline early works on human RNase P as a tRNA processing enzyme and comment on its expanding nonconventional functions in molecular networks of transcription, chromatin remodeling, homology-directed repair, and innate immunity. The important implications and insights from these discoveries on the potential use of RNase P as a gene-targeting tool are presented. This multifunctionality calls to a modified structure-function partitioning of domains in human RNase P, as well as its relative ribonucleoprotein, RNase MRP. The role of these two catalysts in innate immunity is of particular interest in molecular evolution, as this dynamic molecular network could have originated and evolved from primordial enzymes and sensors of RNA, including predecessors of these two ribonucleoproteins.

Gambogic acid and juglone inhibit RNase P through distinct mechanisms.

The first step in transfer RNA (tRNA) maturation is the cleavage of the 5' end of precursor tRNA (pre-tRNA) catalyzed by ribonuclease P (RNase P). RNase P is either a ribonucleoprotein complex with a catalytic RNA subunit or a protein-only RNase P (PRORP). In most land plants, algae, and Euglenozoa, PRORP is a single-subunit enzyme. There are currently no inhibitors of PRORP for use as tools to study the biological function of this enzyme. Therefore, we screened for compounds that inhibit the activity of a model PRORP from A. thaliana organelles (PRORP1) using a high throughput fluorescence polarization cleavage assay. Two compounds, gambogic acid and juglone (5-hydroxy-1,4-naphthalenedione) that inhibit PRORP1 in the 1 µM range were identified and analyzed. We found these compounds similarly inhibit human mtRNase P, a multisubunit protein enzyme and are 50-fold less potent against bacterial RNA-dependent RNase P. Our biochemical measurements indicate that gambogic acid is a rapid-binding, uncompetitive inhibitor targeting the PRORP1-substrate complex, while juglone acts as a time-dependent PRORP1 inhibitor. Additionally, X-ray crystal structures of PRORP1 in complex with juglone demonstrate the formation of a covalent complex with cysteine side chains on the surface of the protein. Finally, we propose a model consistent with the kinetic data that involves juglone binding to PRORP1 rapidly to form an inactive enzyme-inhibitor complex and then undergoing a slow step to form an inactive covalent adduct with PRORP1. These inhibitors have the potential to be developed into tools to probe PRORP structure and function relationships.

Processing of the alaW alaX operon encoding the Ala2 tRNAs in Escherichia coli requires both RNase E and RNase P.

The alaW alaX operon encodes the Ala2 tRNAs, one of the two alanine tRNA isotypes in Escherichia coli. Our previous RNA-seq study showed that alaW alaX dicistronic RNA levels increased significantly in the absence of both RNase P and poly(A) polymerase I (PAP I), suggesting a role of polyadenylation in its stability. In this report, we show that RNase E initiates the processing of the primary alaW alaX precursor RNA by removing the Rho-independent transcription terminator, which appears to be the rate limiting step in the separation and maturation of the Ala2 pre-tRNAs by RNase P. Failure to separate the alaW and alaX pre-tRNAs by RNase P leads to poly(A)-mediated degradation of the dicistronic RNAs by polynucleotide phosphorylase (PNPase) and RNase R. Surprisingly, the thermosensitive RNase E encoded by the rne-1 allele is highly efficient in removing the terminator (>99%) at the nonpermissive temperature suggesting a significant caveat in experiments using this allele. Together, our data present a comprehensive picture of the Ala2 tRNA processing pathway and demonstrate that unprocessed RNase P substrates are degraded via a poly(A) mediated decay pathway.

Structural and mechanistic basis for recognition of alternative tRNA precursor substrates by bacterial ribonuclease P.

Binding of precursor tRNAs (ptRNAs) by bacterial ribonuclease P (RNase P) involves an encounter complex (ES) that isomerizes to a catalytic conformation (ES*). However, the structures of intermediates and the conformational changes that occur during binding are poorly understood. Here, we show that pairing between the 5' leader and 3'RCCA extending the acceptor stem of ptRNA inhibits ES* formation. Cryo-electron microscopy single particle analysis reveals a dynamic enzyme that becomes ordered upon formation of ES* in which extended acceptor stem pairing is unwound. Comparisons of structures with alternative ptRNAs reveals that once unwinding is completed RNase P primarily uses stacking interactions and shape complementarity to accommodate alternative sequences at its cleavage site. Our study reveals active site interactions and conformational changes that drive molecular recognition by RNase P and lays the foundation for understanding how binding interactions are linked to helix unwinding and catalysis.