Functional reconstitution and characterization of
            <i>Pyrococcus furiosus</i>
            RNase P

Tsai, Hui Lien; Pulukkunat, Dileep K.; Woznick, Walter K.; Gopalan, Venkat

doi:10.1073/pnas.0608000103

Cited by 86 publications

(217 citation statements)

References 49 publications

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“…Reconstitution studies have revealed that the archaeal RNase P proteins function as two binary complexes: Pop5• Rpp30 and Rpp21•Rpp29 (23). Footprinting studies indicate that Pop5•Rpp30 interacts with the C domain, and Rpp21• Rpp29 interacts with the S domain of archaeal RNase P RNA (23,28).…”

Section: Resultsmentioning

confidence: 99%

“…S4) with one surprising difference. All known bacterial and archaeal RNase P RNAs consist of both a substrate-specificity (S) domain that aids substrate recognition, and a catalytic (C) domain essential for phosphodiester cleavage (22,23). The Pyrobaculum RNase P RNA candidates have lost most of their S domain, but retain an intact C domain that includes all 11 universally conserved nucleotides (24) (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Discovery of a minimal form of RNase P in Pyrobaculum

Lai

Chan

Cozen

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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RNase P RNA is an ancient, nearly universal feature of life. As part of the ribonucleoprotein RNase P complex, the RNA component catalyzes essential removal of 5′ leaders in pre-tRNAs. In 2004, Li and Altman computationally identified the RNase P RNA gene in all but three sequenced microbes: Nanoarchaeum equitans, Pyrobaculum aerophilum, and Aquifex aeolicus (all hyperthermophiles) [Li Y, Altman S (2004) RNA 10:1533-1540. A recent study concluded that N. equitans does not have or require RNase P activity because it lacks 5′ tRNA leaders. The "missing" RNase P RNAs in the other two species is perplexing given evidence or predictions that tRNAs are trimmed in both, prompting speculation that they may have developed novel alternatives to 5′ pre-tRNA processing. Using comparative genomics and improved computational methods, we have now identified a radically minimized form of the RNase P RNA in five Pyrobaculum species and the related crenarchaea Caldivirga maquilingensis and Vulcanisaeta distributa, all retaining a conventional catalytic domain, but lacking a recognizable specificity domain. We confirmed 5′ tRNA processing activity by high-throughput RNA sequencing and in vitro biochemical assays. The Pyrobaculum and Caldivirga RNase P RNAs are the smallest naturally occurring form yet discovered to function as trans-acting precursor tRNA-processing ribozymes. Loss of the specificity domain in these RNAs suggests altered substrate specificity and could be a useful model for finding other potential roles of RNase P. This study illustrates an effective combination of next-generation RNA sequencing, computational genomics, and biochemistry to identify a divergent, formerly undetectable variant of an essential noncoding RNA gene.catalytic RNA | gene finding | RNA processing R Nase P is best known for its role in removing the 5′ leaders of pre-tRNAs, an essential step in tRNA maturation. It also processes other RNAs in bacteria and eukaryotes, but these roles are less understood (1-3). RNase P typically functions as an RNA-protein complex, comprised of one conserved RNA and a varying number of protein subunits, depending on the domain of life: one in Bacteria, at least four in Archaea, and nine or more in the eukaryotic nucleus (4, 5). A precedent in which the RNA component is missing entirely is found in human and Arabidopsis organellar RNase P (6, 7), although a recent study suggests the possible coexistence of protein-only and RNA-protein-based RNase P complexes in human mitochondria (8).The inability to identify RNase P in some organisms has sown doubts about whether it is a universal feature of life. Studies of the hyperthermophilic bacterium Aquifex aeolicus showed that it exhibits RNase P-like trimming of tRNAs (9, 10), yet a gene for the expected protein component is absent and the RNA has remained elusive (11), prompting speculation that it may have developed a unique solution for pre-tRNA processing (9). Perhaps most surprisingly, Söll and coworkers (12) demonstrated that the archaeal symbiont Nanoarchaeum equ...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Discovery of a minimal form of RNase P in Pyrobaculum

Lai

Chan

Cozen

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…This region is the high-affinity binding site of L7Ae in Pho RPR, and its absence in type M archaeal and all eukaryal RPRs suggests that a universal role for L7Ae and its homologs in RNase P catalysis must involve another RPR region. Therefore, we used a type M archaeal RNase P to investigate if L7Ae is indeed part of the holoenzyme and to understand how it influences catalysis.Using purified recombinant subunits, we and others have reconstituted type A and M RNase P from different thermophilic archaeal variants [Mth, Pho, Pyrococcus furiosus (Pfu) and Methanocaldococcus jannaschii (Mja)] (13,15,25,26). Because we expected studies on a mesophilic type M variant to yield results that are also applicable to eukaryal RNase P, we decided to focus on RNase P from Methanococcus maripaludis (Mma); this choice Author contributions: I-M.C…”

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confidence: 99%

“…Using purified recombinant subunits, we and others have reconstituted type A and M RNase P from different thermophilic archaeal variants [Mth, Pho, Pyrococcus furiosus (Pfu) and Methanocaldococcus jannaschii (Mja)] (13,15,25,26). Because we expected studies on a mesophilic type M variant to yield results that are also applicable to eukaryal RNase P, we decided to focus on RNase P from Methanococcus maripaludis (Mma); this choice was also inspired in part by the fact that transformation and homologous recombination are possible for Mma (27,28).…”

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Ribosomal protein L7Ae is a subunit of archaeal RNase P

Cho

Lai

Susanti

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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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 k cat ∕K m (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.pre-tRNA processing | RPP38 | protein-aided RNA catalysis R Nase P is a Mg 2þ -dependent endoribonuclease that is primarily responsible for catalyzing the removal of the 5′ leaders of precursor-tRNAs (pre-tRNAs) (1-3). Except for some unique organellar variants, RNase P functions in all three domains of life as a ribonucleoprotein (RNP) (1, 2). Although catalysis rests with the essential RNase P RNA (RPR) in all three domains of life (4-6), the RNase P protein (RPP) cofactors play essential roles. In the simple one RPR-one RPP bacterial RNase P, the RPP aids RPR catalysis by enhancing cleavage efficiency and affinity for substrate and Mg 2þ (7-9). The bacterial RPP has not been found in any archaeal or eukaryal genome (10). Eukaryal (nuclear) RNase P, which comprises an RPR and 9 or 10 RPPs (11, 12), has not been reconstituted from recombinant subunits, thus thwarting efforts to uncover the individual functions of eukaryal RPPs. Archaeal RNase P, with an RPR and four RPPs (all homologous to eukaryal RPPs), has therefore been explored as an experimental surrogate for its so-far-intractable eukaryal cousin (13-16). Although native archaeal RNase P has not been characterized, Western analysis and immunoprecipitation validated these four RPPs (POP5, RPP21, RPP29, and RPP30) as being associated with partially purified Methanothermobacter thermautotrophicus (Mth) RNase P activity (14). Subsequent structural and biochemical reconstitution studies using recombinant subunits have proven the utility of archaeal RNase P as a model system to dissect the role of multiple protein cofactors in facilitating RNA catalysis (16).Besides POP5, RPP21, RPP29, and RPP30, weak homologies are evident in the archaeal genomes...

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“…First, reproducing the results of Kikovska et al (9) with different archaeal/eukaryal RPRs will bolster the idea that the catalytic core really rests with the RPR during evolution of RNase P. The corollary is that Rpps facilitate RPR catalysis by fine-tuning/stabilizing the active site for productive positioning of the substrate and catalytically important metal ions. Second, having conditions under which the human RPR is weakly active will permit an evaluation of the functional roles of individual human Rpps, as demonstrated for archaeal RNase P (15,16). Third, if eukaryal RPRs do form shortlived functional structures, cross-linking or RNA engineering could be attempted to trap these conformations and demonstrate that such RNAs could be weaned of their dependence on some Rpps.…”

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confidence: 99%