From the Department of Microbiology and the College
of Medicine, University of Illinois at Urbana-Champaign, Chemical and
Life Sciences Laboratory, Urbana, Illinois 61801
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ABSTRACT |
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In eukaryotes, ribonuclease P (RNase P) requires both RNA and protein components for catalytic activity. The eukaryotic RNase P RNA, unlike its bacterial counterparts, does not possess intrinsic catalytic activity in the absence of holoenzyme protein components. We have used a sensitive photoreactive cross-linking assay to explore the substrate-binding environment for different eukaryotic RNase P holoenzymes. Protein components from the Tetrahymena thermophila and human RNase P holoenzymes form specific products in photoreactions containing [4-thio]-uridine-labeled pre-tRNAGln. The HeLa RNase P RNA in neither the presence nor the absence of holoenzyme protein components formed cross-link products to the pre-tRNAGln probe. Parallel photo-cross-linking experiments with the Escherichia coli RNase P holoenzyme revealed that only the bacterial RNase P RNA forms specific substrate photoadducts. A protein-rich active site for the eukaryotic RNase P represents one unique feature that distinguishes holoenzyme organization between bacteria and eukaryotes.
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INTRODUCTION |
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Cleavage of nascent tRNA transcripts to generate the mature 5' termini is catalyzed by the ribonucleoprotein complex ribonuclease P (RNase P)1 (1). A key point yet to be addressed in RNase P function is whether all RNase P holoenzymes share a common catalytic center. Although all RNase P holoenzymes require both RNA and protein components for function, only the bacterial RNase P RNA is catalytically active in the absence of its protein complement (2). The proposed secondary structures of bacterial and eukaryotic RNase P RNAs are similar (3), implying that eukaryotic RNase P RNA, like its bacterial counterparts, plays a central role in pre-tRNA processing.
Substrate cross-linking methodologies provide an informative means whereby one can establish the identity of interacting active site components (4). This report describes the use of a photoreactive pre-tRNA substrate to characterize the composition of the eukaryotic RNase P active site. We identified one protein component within the Tetrahymena thermophila holoenzyme that lies in proximity to the substrate when probed under optimal pre-tRNA processing reaction conditions. We extended these studies to the RNase P RNA and to the holoenzymes isolated from the human HeLa cell line and Escherichia coli. In eukaryotic examples of RNase P, protein components in the holoenzyme, rather than RNA, were found to form cross-link products to the photoreactive substrate. These findings provide evidence that proteins of eukaryotic RNase P holoenzymes constitute significant components of the active site architecture.
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EXPERIMENTAL PROCEDURES |
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Preparation of Ribonuclease P Holoenzymes and T7 RNA Polymerase-- The RNase P from T. thermophila was partially purified to ~800-fold enrichment using our previously published procedure (5). The Tetrahymena cells were harvested and washed with water at room temperature to reduce premature lysis and release of proteases.2 The RNase P from HeLa cells was partially purified ~100-fold using two successive rounds of DEAE-Sepharose chromatography and glycerol gradient centrifugation (6, 7). The RNase P from E. coli strain SG22094 (8) was partially purified using DEAE-Sephadex A-50 chromatography, precipitation of RNase P from peak activity fractions with ammonium sulfate, and gel filtration chromatography with Sepharose CL-4B (9). Expression and purification of a recombinant, hexahistidine-tagged E. coli RNase P protein was performed using Nickel(II)-nitrilo-triacetic acid chromatography (10). The reconstitution of E. coli holoenzyme using purified components was carried out by the method of slow dialysis (9). RNase P processing assays were performed to verify that enzymatic activity was restored to the E. coli holoenzyme following reconstitution (10). T7 RNA polymerase was purified to >95% homogeneity from E. coli BL21[pAR1219] (11) using SP-Sepharose High Performance resin chromatography and Q-Sepharose Fast Flow resin chromatography.
Preparation of RNA Transcripts-- The transcription template for the preparation of the HeLa RNase P RNA was generated using gene-specific primers to the HeLa RNase P RNA coding sequence, total RNA prepared from the HeLa S3 cell line, and a reverse transcription-polymerase chain reaction method (12). First strand cDNA synthesis was done using SuperscriptTM II reverse transcriptase (Life Technologies, Inc.), and DNA amplification was accomplished using VentTM DNA polymerase (New England Biolabs). The gene-specific oligonucleotide primers (Operon, Inc.) have the following sequences: H1-5'PRNA, 5'-GCTCTAGATAATACGACTCACTATAGGATAGGGCGGAGGGAAGCTCATC-3', and H1-3'PRNA, 5'-CGGAATTCTCTTCTAATGGGCGGAGGAGAGTAG-3'. The amplified product was cleaved with EcoRI and XbaI restriction endonucleases (New England Biolabs) and introduced into pSP65 (Promega) to generate pSP65-T7H1. The resultant plasmid contains a T7 RNA polymerase promoter adjacent to the first nucleotide of the RNase P RNA coding sequence and a unique Ksp632I site adjacent to the last nucleotide of the RNase P RNA coding sequence. The RNase P RNA transcript was generated from this template using T7 RNA polymerase following Ksp632I (Boehringer Mannheim) cleavage of the plasmid. The bacterial RNase P RNA was transcribed in vitro using T7 RNA polymerase as described previously (13). The RNA transcripts were purified from a denaturing gel, and the RNAs were refolded by heating to 80 °C for 3 min and adding buffer components containing MgCl2 just prior to cooling.
The Tetrahymena pre-tRNAGln substrate was transcribed using T7 RNA polymerase as described previously (5). The photoreactive substrate was transcribed with [4-thio]-UTP (Amersham Life Science) present in the reaction mixture to allow the incorporation of [4-thio]-UMP into the transcript (4). The substrate RNA was transcribed and purified under reduced light conditions. All subsequent manipulations with the RNA were performed in amber-colored tubes (Fisher) and under reduced light conditions to prevent premature photolysis of the substrate.Photochemical Cross-linking Experiments-- The refolded substrate (0.50 pmol) was incubated with RNase P holoenzyme (~1 µg) in a final volume of 20 µl under optimal reaction conditions at 37 °C for 20 min to allow the enzyme to bind to the substrate. The optimal pre-tRNA processing reaction conditions differ for each of the tested holoenzymes. The cross-linking experiments with a given holoenzyme were performed under buffer conditions that support optimal pre-tRNA processing activity for that holoenzyme. The following reaction conditions were used: T. thermophila RNase P: 50 mM Tris-HCl (pH 7.5), 7.5 mM MgCl2 (5); HeLa RNase P: 50 mM Tris-HCl (pH 7.5), 100 mM NH4Cl, 10 mM MgCl2 (6); and E. coli RNase P: 50 mM Tris-HCl (pH 8.0), 100 mM NH4Cl, 15 mM MgCl2 (14).
The cross-linking experiments were performed with the RNase P RNA transcripts at a final concentration of 200 nM in 20 µl of buffer C (50 mM Tris-HCl (pH 7.6), 100 mM NH4Cl, 60 mM MgCl2) (2). The cross-linking studies with HeLa RNase P RNA were performed under several additional buffer conditions besides buffer C. These buffer conditions included variations in monovalent salt concentration (0.05-1.0 M NH4Cl or KCl), MgCl2 concentration (0.01-0.10 M), and spermidine concentration (0-20 mM). All mixtures were irradiated on ice through a plastic Petri dish filter with 254 nm ultraviolet light from a UVP model UVG-11 lamp at an approximate distance of 2 cm for 1 h. Each mixture was recovered and placed in an amber tube for further enzymatic manipulations. When analyzing RNA-substrate cross-link products from holoenzymes, self-digested Pronase (125 µg) was added, and the reaction was incubated at 37 °C for 1 h. Self-digested Pronase is nuclease-free, as judged by the inability of this enzyme to degrade the E. coli RNase P RNA subunit or the pre-tRNAGln probe used in this study. RNA-specific cross-link products were analyzed by denaturing gel electrophoresis using urea (8 M)-polyacrylamide (6%) gel systems. The gels were dried and subjected to autoradiography. When analyzing protein-substrate cross-link products, RNase T1 (10 units) was added, and the reaction was incubated at 37 °C for 1 h. Protein-specific cross-link products were analyzed by gel electrophoresis using SDS-polyacrylamide (10%) gel systems. The gels were fixed, dried, and subjected to autoradiography. The formation of specific photo-cross-link products was found to be linear with time and varied proportionately with the amount of holoenzyme components present.Immunological Reagents and Methods--
Anti-RNase P polyclonal
serum was generated in the Immunological Resource Center of the
University of Illinois. BALB/c mice were injected intraperitoneally
with purified Tetrahymena RNase P holoenzyme mixed with
Freund's complete adjuvant (Difco) for the primary immunization and
with Freund's incomplete adjuvant (Difco) for all subsequent
immunizations. The presence of anti-RNase P antibodies was determined
by immunodepletion studies of RNase P activity. Ascites fluid
production of the antisera was accomplished by intraperitoneal
injection of 106 Sp2/0-Ag14 myeloma cells per mouse
following pristane treatment of the immunized animals (15). The IgG was
purified from an enriched, delipidfied -globulin fraction of the
serum using protein A-Sepharose 4B resin (16).
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RESULTS |
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The Tetrahymena holoenzyme displays a
Km of ~160 nM for the homologous
pre-tRNAGln, which is typical for RNase P enzymes (5). We
prepared an internally labeled photoreactive substrate probe using this
pre-tRNA to explore the molecular composition of the active site for
the Tetrahymena holoenzyme. The pre-tRNAGln
contains [4-thio]-uridine, a nucleoside analog that promotes efficient formation of photoadducts upon exposure to UV light (4). This
substrate also has internal -32P-adenosine to facilitate
the detection of photoadducts. The pre-tRNAGln probe is
readily cleaved in the dark by RNase P preparations obtained from
T. thermophila, indicating that the presence of the
[4-thio]-uridine in the transcript does not inhibit processing activity (not shown).
Irradiation of enzyme-substrate mixtures with UV light resulted in the formation of cross-link products. The nature of the cross-linked species was initially analyzed by treating aliquots of the mixture with self-digested Pronase to remove any proteins present. The Pronase-treated cross-linked mixtures were fractionated on denaturing polyacrylamide gels to analyze RNase P RNA-substrate RNA cross-link products, but no RNA-substrate adducts were evident. The Pronase preparation used in these experiments was analyzed for its ability to degrade RNA. The E. coli RNase P RNA and pre-tRNAGln were not degraded when they were individually treated with the Pronase preparation used in these experiments (not shown).
The holoenzyme-substrate cross-linked mixtures were treated with RNase T1 to remove any RNA not associated with protein. Nuclease-treated reaction mixtures were fractionated on SDS-PAGE systems, and one specific polypeptide-substrate adduct was consistently observed (Fig. 1, lanes 3 and 4). The intensity of the Tetrahymena photoproduct correlated with the abundance of RNase P activity observed throughout purification. This product is eliminated with incubation of the cross-linked holoenzyme mixture with Pronase or proteinase K (not shown). The generation of this photoadduct required the presence of the photoreactive nucleoside analog in the substrate transcript (not shown), the presence of RNase P holoenzyme, and exposure to UV light (Fig. 1, lane 5).
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The estimated size of the polypeptide in the Tetrahymena photoadduct formed is ~36 kDa. Purification efforts with the T. thermophila RNase P resulted in the enrichment of a 36-kDa protein, as shown in Fig. 2A. A mouse polyclonal antiserum generated against partially purified RNase P contains antibodies that recognize this 36-kDa protein, as demonstrated by immunoblot analysis (Fig. 2A, lower panel). These antibodies were used to immunodeplete T. thermophila RNase P activity from an extract, whereas preimmune (normal) mouse antiserum did not immunodeplete the activity (Fig. 2B). This antiserum also immunoprecipitated the polypeptide photoadduct following nuclease digestion of the mixture (Fig. 2C), thereby providing evidence that this cross-link product comprises a protein component from RNase P.
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We next tested whether proteins cross-link to the substrate in two other well characterized RNase P holoenzymes. Photocross-linking assays were performed with two different preparations of E. coli RNase P holoenzymes, one partially purified from cell lysates and another derived from in vitro reconstitution from individual components. No cross-link products were generated between the substrate probe and the bacterial holoenzyme protein component. Several proteins copurify with the human RNase P enzyme; one predominant polypeptide of ~115 kDa and two smaller proteins formed photoadducts with the pre-tRNAGln probe when the cross-linking reaction was performed under pre-tRNA processing reaction conditions (Fig. 1, lane 1). The two smaller cross-linked species are not present in parallel cross-linking experiments with a repeated purification of this RNase P holoenzyme, suggesting that these cross-linked species are either unrelated to RNase P or may have been degradation products. The 115-kDa cross-link product is lost following treatment of cross-linked holoenzyme mixtures with Pronase (not shown). The intensity of the 115-kDa cross-link product correlates with the abundance of RNase P activity from HeLa cell lysates and also requires exposure to UV light (Fig. 1, lane 2). This protein is similar in size to the previously identified hPop1 protein that is an ortholog to the Saccharomyces cerevisiae Pop1p (19).
The lack of Pronase-resistant cross-link products generated with the Tetrahymena holoenzyme prompted us to examine whether the pre-tRNAGln probe could generate a cross-link product with any RNase P RNA. The E. coli RNase P RNA has been shown previously to bind 5'-[4-azidophenacyl]-tRNAPhe and form specific photoadducts with that probe (18).3 We incubated the bacterial RNase P RNA with the [4-thio]-uridine pre-tRNAGln probe under optimal catalytic reaction conditions prior to irradiation. The E. coli RNase P RNA efficiently cleaved the substrate probe to generate mature tRNAGln and 5'-leader RNA products of the processing reaction (not shown). The E. coli RNase P RNA formed specific cross-link species with the pre-tRNAGln substrate (Fig. 3). We conclude that the pre-tRNAGln probe harbors sufficient photoreactivity with RNA to enable the detection of RNA-RNA adducts in these studies.
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We also did not detect Pronase-resistant cross-link products when the HeLa holoenzyme was incubated with the pre-tRNAGln probe (not shown). Although the proposed secondary structure for the HeLa RNase P RNA resembles that of the bacterial RNase P RNA (3), this molecule has not been demonstrated to harbor catalytic activity (6). The HeLa RNase P RNA was transcribed in vitro, purified, refolded, and analyzed directly for the ability to bind substrate RNA using photochemical cross-linking assays with [4-thio]-uridine-labeled pre-tRNAGln. The refolded RNase P RNA was incubated with the photoreactive pre-tRNAGln prior to irradiation. The HeLa RNase P RNA did not generate any cross-link products with pre-tRNAGln under a variety of reaction conditions tested that included variations in monovalent salt concentration, MgCl2 concentration, and spermidine concentration (Fig. 3, lane 4, and data not shown).4
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DISCUSSION |
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The lack of demonstrable catalytic activity for an isolated eukaryotic RNase P RNA argues that the functional role of the protein complement of eukaryotic RNase P is more substantial than that of its bacterial counterpart. The eukaryotic protein subunit(s) may be involved in RNA folding, compartmentalization, or some more direct aspect of the catalytic cycle. The results presented in this report reveal that protein components of eukaryotic RNase P are in proximity to the pre-tRNA substrate. Cross-linking experiments that were performed earlier to identify components of the Schizosacchromyces pombe RNase P holoenzyme revealed that a polypeptide of approximately 100 kDa forms a photoadduct with a substrate affinity probe (20). The S. cerevisiae RNase P forms a specific complex with pre-tRNA in a native gel system following extensive micrococcal nuclease digestion of the RNA component of the holoenzyme (21). These findings reinforce the notion that the protein complement of eukaryotic RNase P holoenzymes contribute to the active site structure.
One role of active site components in eukaryotic RNase P function is to bind to pre-tRNA substrates and position them for correct cleavage at the 5' terminus. Cleavage of pre-tRNA may be mediated by the eukaryotic RNA component, which is required for activity of all holoenzymes under physiological conditions. The inability of the eukaryotic RNase P RNAs to efficiently bind substrate may account for their lack of catalytic activity in RNA-only reactions. The prevalence of protein components near the pre-tRNA suggests that active site-associated proteins may contribute to substrate binding functions during the catalytic cycle.
One phylogenetic analysis of bacterial and eukaryotic P RNAs has led to the identification of a distinct region of the bacterial RNase P RNA (P15) that is absent in eukaryotic RNase P RNA (3). This region of bacterial RNase P RNA is critical for tRNA recognition and binding (22). Coupled with the cross-linking data suggesting that proteins are prevalent in the active site architecture, these results suggest that during evolution a switch in the molecular composition of the substrate-binding domain occurred where it may have gradually shifted from one composed of RNA to one composed of protein. The functional hand-off envisioned from the bacterial RNase P RNA to the eukaryotic protein complement within RNase P is a feature observed in another example of a catalytic ribonucleoprotein complex. The cyt-18 gene product potentiates catalytic RNA function for group I intron RNA molecules that lack endogenous stabilizing domains (23, 24). The rationale for such functional reassignments among various holoenzyme components is not yet understood. The dramatic reconfiguration in active site architecture found for RNase P and group I introns may be more prevalent in nature as remnants of an RNA world resurface in the world dominated by protein catalysts.
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ACKNOWLEDGEMENTS |
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We thank Drs. D. Allis and D. Engelke for helpful discussions; Drs. T. Cech, N. Pace, and R. Rivera-León for providing reagents used in this work; and A. Gooding for sharing the unpublished T7 RNA polymerase purification procedure.
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FOOTNOTES |
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* This work was supported in part by Grant GM47854 from the U.S. Public Health Service and a University of Illinois campus research board award.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Microbiology, University of Illinois at Urbana-Champaign, B103 Chemical and Life Sciences Lab., 601 South Goodwin Ave., Urbana, IL 61801. Tel.: 217-244-6433; Fax: 217-244-6433; E-mail: dcelande{at}uiuc.edu.
1 The abbreviations used are: RNase P, ribonuclease P; PAGE, polyacrylamide gel electrophoresis.
2 D. Allis, personal communication.
3 The 5'-[4-azidophenacyl] tRNAPhe substrate was synthesized according to the method described previously (18) and used in photo-cross-linking assays with E. coli RNase P RNA. The pattern of E. coli RNase P RNA-tRNA adducts obtained following irradiation was similar to that observed previously (Ref. 18 and data not shown).
4 The HeLa RNase P RNA also failed to yield cross-link photoadducts when incubated alone with a different photoreactive substrate analog, 5'-[4-azidophenacyl] tRNAPhe (data not shown).
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REFERENCES |
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