The RNA Component of RNase P from the Archaebacterium

THE JOURNAL OF BIOLOGICAL CHEMISTRY (c’ 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 9, Issue of March 25, pp. 5689-5695, 1991 Printed in 11. S. A.

The RNA Component of RNase P from the Archaebacterium Haloferax volcanii”

(Received for publication, September 19, 1990)

Daniel T. NieuwlandtS, Elizabeth S. Haas$, and Charles J. DanielsSll From the $Department of Microbiology, Ohio State University, Columbus, Ohio 43210 and the §Department of Biology, Indiana University, Bloomington, Indiana 47405

RNase P, an endoribonuclease responsible for gen- erating the mature 5‘ termini of tRNA precursors, is composed of both RNA and protein. It has been dem- onstrated that the eubacterial RNase P RNA will, under the appropriate reaction conditions, exhibit catalytic activity in vitro. Evidence has not been obtained for catalytic activity by the RNAs of eukaryotic RNase P enzymes. Using a cDNA probe prepared from RNA copurifying with RNase P activity from the archaebacterium Haloferax volcanii, we have characterized the gene encoding the RNase P RNA. The proposed transcript from this gene can assume a structure resem- bling the eubacterial RNase P RNA and includes many of the highly conserved sequences of these RNAs. This RNA was incapable of cleaving pre-tRNA substrates in the absence of protein under a variety of in vitro conditions. Catalytic activity was observed when this RNA was combined with the protein subunit of the Bacillus subtilis RNase P complex. Similarities among the archaebacterial, eubacterial, and eukaryotic RNase P RNA sequences and structures are discussed.

Archaebacteria, like the eubacteria and eukaryotes, possess an activity that removes the 5’ leader sequences from tRNA primary transcripts. In eubacterial and eukaryotic cells, this reaction is carried out by the enzyme RNase P, which is composed of both protein and RNA (for reviews, see Refs. 1- 3) . The role of the RNA component in the eubacterial RNase P complex is now clear; RNA functions as the catalytic component (4, 5 ) . Despite the common feature of RNA catalysis shared by these enzymes, sequence analysis has shown that there is considerable divergence between these RNAs. The RNase P RNAs from Escherichia coli and Bacillus subtilis exhibit only 43% sequence similarity (11). Recently, a common feature has been observed that again relates these diver- gent RNAs. A comparison of a phylogenetically diverse group of eubacteria has shown that these RNAs share a common structural core, where conserved nucleotides are localized to similar structural regions (12, 13). While evidence suggests that some eukaryotic RNase P complexes require RNA, there

*This work was supported by Office of Naval Research Grant N00014-89-5-3077. The costs of publication of this article were de- frayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide .sequence(.$ reported in this paper has been submitted

M6 1003. to the GenBank””/EMBL Data Bank with accession number&

n Associate of the Canadian Institutes for Advanced Research. To whom correspondence should be sent: Dept. of Microbiology, Ohio State University, 484 W. 12th Ave., Columbus, OH 43210. Tel.: 614. 292-4599; Fax: 614-292-1538.

is no direct evidence that the RNA itself is capable of cleaving tRNA precursors (6-10). In addition, these RNAs bear little sequence similarity to their eubacterial counterparts and lack the structural core characteristic of these RNAs.

Differences in the structure and catalytic capabilities of the RNase P RNAs from the eubacteria and eukaryotes bring to question the nature of archaebacterial RNase P enzymes. As representatives of a third evolutionary line of descent (14), these organisms contain a mosaic of molecular characteristics. Some of their molecular features are eubacterial-like, whereas others are eukaryotic-like. For example, they have rRNA operons and operon structures in general similar to the eubacteria. Contrasting this, some stable RNA genes are inter- rupted by introns. Other genes, like those encoding ribosomal proteins and RNA polymerases, encode proteins that are eukaryotic in nature (15).

Two reports have described the occurrence of RNase P activity in the archaebacteria. RNase P from the halophilic archaebacterium, Haloferar uolcanii, has been reported to be micrococcal nuclease-sensitive and to have a buoyant density of 1.61 g/cm” in cesium sulfate (16). These data are consistent with the proposal that this enzyme has a required RNA component and suggest that, like the eubacterial complex, it is composed of a large RNA and a small protein. A second report described the RNase P activity from Sulfolobus solfa- taricus, a member of the acid thermophile branch of the archaebacteria (17). This enzyme was not sensitive to micrococcal nuclease and had a buoyant density of 1.27 g/cm:‘ in cesium sulfate. This apparent higher protein:nucleic acid ratio resembles the eukaryotic and organellar RNase P activities. These studies suggested a diversity among the archaebacterial RNase P enzymes and left unresolved the role of RNA in catalysis.

In this report, we describe the isolation of a gene encoding an RNA that copurifies with H. uolcanii RNase P activity. The transcript from this gene can assume a folded structure that is similar to eubacterial RNase P RNA structures and shares many of the highly conserved sequences of these RNAs. Under a variety of i n vitro incubation conditions, this RNA was incapable of catalyzing cleavage of pre-tRNAs. However, catalytic activity was observed when this RNA was combined with B. subtilis RNase P protein.

MATERIALS AND METHODS’

RESULTS

Strong evidence has been obtained for the presence of an RNA component in the RNase P of H. uolcanii prepared from

’ Portions of this paper (including “Materials and Methods” and Figs. 1, 2, and 7) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

5689

5690 RNase P RNA from an Archaebacterium

FIG. 3. Sequence of the H. uolcanii RNase P RNA gene region. Underlined sequences represent the RNase P RNA. Arrows indicate possible hairpins, and potential transcription sig- nals (Box A and Box B) are indicated. Plasmid pDN3, used in the synthesis of RNA for in vitro RNA catalysis assays, was constructed from the indicated MaeI-BstBI fragment. An oligonucleotide complementary to the sequences indicated by the heavy bar was used in primer extension analysis and for the synthesis of DNA probes in S1 mapping.

G C G G C l C G ~ G R R R C A G C C R G C l C G G C ~ ~ ~ R G C G C G G C R O C C C l l l G C G ~ G ~ ~ C l G G

G l T C G C C G C C G C ~ G T C T C G G l ~ l G C G C G G C l l G ~ C l C l C C l C G l G G C G G l G C ~ C ~ l C G l C 840

CRUGCGGCGGCGTCRGRGCCRTRCGCGCCG~~ClG~G~GGUGC~CCGCC~CG~Gl~GC~G

C T C G C C G G C l G T G C C G C G C C G G l G l C G C C G G G ~ ~ C C G R C G G G ~ C G ~ R G C G ~ C G ~ C l G C C 900

GRGCGGCCGRCRCGGCGCGGCCRCRGCGGCCCllGGClGCCClGClGlCGCTGClG~CGG

UCGTClGC~GClTCGCCGCCGRCCRetGRCTCOGCRRCCGGC~~CCG~GlCGCCCGCGGCG~CCGCG 960

1GCRGACGCCGRRGCGGCGGClGGlGGCTG~GCCGllGGClC~GCGGGCGCCGClGGCGC

c

TGGGGClGCTGRRGCGGRGGC~GClG RECCCGRCGRCTTCGCCTCCGTrGRC

b l I

low salt extracts (16). This activity is sensitive to micrococcal nuclease and has a high buoyant density (1.61 g/cm3). We have observed that fractionation of extracts from these cells by gel filtration on Sepharose 4B followed by Sephadex G- 200 in 2 M KC1 (physiological salt conditions) gave an enzyme that remained active over a broad range of salt conditions (0.05-3 M KCl; data not shown). Consistent with the earlier observation that this enzyme contains an RNA component, we also noted that an approximately 435-nucleotide RNA copurified with this activity.

To determine if this RNA was a component of the RNase P activity of these cells, the gene encoding this RNA was cloned. cDNAs were prepared from a narrow window of RNAs, containing the 435-nucleotide RNA, which had been isolated from RNase P-active Sephadex G-200 fractions. These cDNAs hybridized to a number of restriction fragments, including several containing the genes for rRNAs (Fig. 2). Based on previous hybridization information for the rRNA operons of H. uolcanii (18, 19) and by blocking Southern blots with rDNA, single non-rRNA gene-containing MluI (2.6 kb)' and Sal1 (1.0 kb) restriction fragments that hybridized to the cDNAs were identified (Fig. 2). In parallel, a cosmid bank from H. uolcanii was screened for hybridization with the cDNAs. Again, after eliminating rRNA-containing cosmids, a single cosmid was identified that hybridized to the cDNAs. This cosmid, cos228, was found to contain both the 2.6-kb MluI and 1.0-kb Sal1 fragments. A 985-bp MluI-Sal1 fragment containing the hybridizing region was subcloned and subjected to DNA sequence analysis. The sequence of this fragment is presented in Fig. 3.

To verify that this region encoded an RNA, Northern analysis was performed, using as probes the 2.6-kb MluI- and 985-bp MluI-Sal1 fragments from the cosmid. The oligonucleotide, 5'-NNNGGACTTTCCTCNNC-3' (where N is any nucleotide), which contains sequences complementary to the highly conserved eubacterial RNase P RNA sequence 5'- GAGGAAAGUCC-3' present within the 985-bp MluI-Sal1 fragment was also used as a probe (see Fig. 3). In each case, the DNAs hybridized to a single RNA species of 435 nucleotides. Fig. 4 illustrates the hybridization obtained with the oligonucleotide probe. The 5' end of this RNA was localized by primer extension and S1 mapping to one of 2 G residues located immediately downstream from a region containing four archaebacterial promoter-like sequences (Figs. 3 and 4). Although the 3' terminus of this RNA was not mapped, Northern data and localization of the 5' terminus suggest

'L The abbreviations used are: kb, kilobase(s); bp, base pair(s).

that the transcript ends in a short stretch of U residues, similar to other archaebacterial transcripts.

Using the criteria established from phylogenetic comparisons of eubacterial RNase P RNAs, this RNA can be folded into a structure that is similar to the proposed eubacterial structure (Fig. 5). The H. uolcanii RNA can assume a three- loop core structure with base-pairing interactions between the 5' and 3' ends; it also has several of the conserved helical structures that protrude from these loops. This similarity extends beyond structural features. The halobacterial RNA contains many of the universally conserved sequence elements of the eubacterial RNase P RNAs, including the longest conserved block, 5'-GAGGAAAGUCC-3' (Fig. 5).

There is little sequence and structure similarity between eubacterial and eukaryotic RNase P RNAs. A small set of nucleotides have been identified in several eukaryotic RNase P RNAs (26); many of these sequences are also present in the halophilic RNase P RNA (Fig. 5). One structural feature that may be conserved between the eubacterial and eukaryotic RNase P RNAs is the formation of a pseudoknot involving two regions of high sequence conservation (13, 27). The halobacterial RNase P RNA also retains the ability to form this pseudoknot structure (Fig. 5).

A fundamental difference between the eubacterial and eukaryotic RNase P RNAs is the ability of the eubacterial RNA to catalyze the cleavage of pre-tRNAs in the absence of protein (4,5). To examine whether the halophilic RNA could act as a catalytic RNA, a MaeI-BstBI fragment containing the gene region was subcloned into the T7 RNA polymerase expression vector pIBI31. Transcripts from this clone produce an RNA that, based on the proposed gene structure, has 20 and 9 nucleotides of 5'- and 3'-flanking sequence, respec- tively. This RNA was assayed with both the halophilic tRNAV"' substrate and the CCA-containing B. subtilis tRNAAap substrate under a variety of solution conditions. Ionic conditions optimal for catalysis by E. coli RNase P RNA (100 mM MgClz and 100 mM NH,Cl) (28) and B. subtilis RNase P RNA (300 mM MgC12 and 1.2 M NH4C1) (29) were tested. Also examined were NH4Cl concentrations from 0.1 to 3.0 M in the presence of 30 mM MgC12, MgClt concentrations from 10 to 250 mM in the presence of 600 mM NH4C1, and KC1 concentrations up to 2 M. Other solution conditions tested were polyethylene glycol 8000, ethanol, and glycerol from 2.5 to 10% and temperatures from 16 to 70 "C in buffer containing 30 mM MgClZ and 1.2 M NH4C1. The halophilic RNA lacked catalytic activity under all conditions tested. As a further test for catalytic activity, the halophilic RNA was

RNase P RNA from an Archaebacterium 5691 1 2 T C C A P L . T C G A S 1

-133 nt

RNA

435 nt- -462 nt

-191 nt

13 C e e

FIG. 4. Transcript analysis of the H. uolcanii RNase P gene. Left panel, Northern analysis of H. uolcanii total RNA using an oligonucleotide probe. This probe contains sequences complementary to a highly conserved eubacterial RNase P sequence that is also present in the 985-bp MluI-Sal1 clone. For center (primer extension (PE) analysis) and right (SI analysis) panels, sequence lanes are presented as the complement of the DNA-coding sequence. Italic segwnces indicate potential transcription start sites. The same DNA primer was used for primer extension synthesis, synthesis of the single strand SI probe, and as primer for DNA sequence markers (see “Materials and Methods” and Fig. 3). Sizes are given as nucleotides (nt).

combined with the protein component of B. subtilis RNase P in a heterologous reconstitution experiment. Incubation conditions were optimized for the activity of the B. subtilis RNase P RNA plus its protein. Under these conditions, the halophilic RNA exhibited cleavage activity with the tRNAVa’ substrate (Fig. 6). Full recovery of the activity was not possible with the heterologous complex. Approximately 10% of the cleavage activity was recovered when compared to the halophilic holoenzyme. Similar results were obtained with the B. subtilis tRNAAsp substrate (Fig. 7).

DISCUSSION

Using cDNAs prepared against RNAs that copurified with RNase P activity from H. volcanii, we have isolated the gene that encodes the RNA component of this enzyme. Identifica- tion of this RNA as a component of the RNase P complex stems from three observations. Previous studies on the phys- ical properties and nuclease sensitivity of this enzyme strongly suggested that this activity had a required RNA component (16). Second, sequence data indicate that this RNA can assume a structure similar to the core structure proposed for eubacterial RNase P RNAs, and it contains many of the conserved sequence elements. And finally, in vitro transcripts from this gene, when combined with the RNase P protein from B. subtilis, exhibit catalytic activity.

A comparison of the structure and sequence features of the halophilic, eubacterial, and eukaryotic RNase P RNAs indi- cates that the halophilic RNA is most similar to the eubacterial RNase P RNA. Its overall structure closely resembles the eubacterial core structure, and it contains 46 of the 60 conserved eubacterial RNase P nucleotides. Further correla- tions between these RNAs with respect to catalytic centers and protein-binding sites are tentative due to the lack of understanding of these issues in the eubacterial enzyme. Some structural regions and specific nucleotides of the RNase P RNA, which have been ascribed to a particular function, have correlates in the halophilic RNA. For example, the protein components of E. coli and B. subtilis RNase P RNAs are thought to interact with sequences in the uppermost loop and the helical region that separates this loop from the central loop (see Fig. 5). In E. coli, the C5 protein protects regions 82-96 and 170-185 (2, 30). A point mutation in this region, GS9 to A”’, leads to a defect in protein association (31).

Insertions into the analogous helical region separating the central and uppermost loops in the B. subtilis RNase P RNA also affect protein interaction (32). The halophilic RNA contains both similar helical structures and related sequences in this region. Therefore, this region of the halophilic RNA may play a role in protein interaction. Other mutations that affect cleavage efficiency in the eubacterial RNase P RNAs have been described. These mutations are located throughout the molecule, and it has been difficult to distinguish “true” catalytic mutations from those that have an indirect effect on the activity due to changes in RNA folding (1, 2). The diversity of structures among the eukaryotic nuclear and organellar RNase P RNAs makes comparisons between these and the other RNAs difficult. One common structural feature that may be shared between the halobacterial, eubacterial, and eukaryotic RNase P RNAs and the mitochondrial RNA processing RNase, is the formation of a pseudoknot (13, 27). Interestingly, these interactions bring together most of the universally conserved sequence elements that are present in distant regions of the molecule. Located in this region of the H. uolcanii RNase P RNA are 12 of the 19 nucleotides conserved between this RNA and the eukaryotic RNase P RNAs (26) (see Fig. 5). Sequence homology between the halobacteria and eukaryotic RNase P RNAs can be extended further if sequences conserved in the pseudoknot interaction are also included. Each eukaryotic RNase P RNA contains the conserved sequence element 5’-GNAANNUCNGNG-3’, which pairs with the sequences of the 3”terminal loop (27) (see Fig. 5). Five of these nucleotides are conserved in the H. uolcanii RNase P RNA. If all of these nucleotides are considered, then 13 nucleotides are conserved in the RNase P RNAs from all three kingdoms. The pseudoknot structure model is also consistent with earlier mutagenesis data for B. subtilis RNase P RNA, which indicated that active site formation involves the interaction between distant portions of the molecule (33).

The inability of the halobacterial RNase P RNA to act catalytically in the absence of protein is puzzling. In the eubacterial system, the protein component appears to act as a cofactor that shields the ionic repulsion forces between the substrate and catalytic RNAs (28,29). Its presence affects the V,,, of the reaction, but not the binding of substrate. It is unlikely that the protein component of the halophilic RNase P plays this role since the internal monovalent ion concentra-

5692 RNase P RNA from an Archebacterium c 5 160

IV

E . GQU

. . . .

IV

Hf. volcanii

IV

HeLa FIG. 5. Structure of the E. coli and H. uolcanii RNase P RNAs. In the upper panel are structures of the RNase P RNAs derived from

phylogenetic comparisons of eubacterial RNase P RNAs (Ref. 13, J. W. Brown, E. S. Haas, and N. R. Pace, personal communications). Circled nucleotides represent nucleotides that are present in similar locations in all eubacterial RNase P RNAs. Arrows in the H . uolcanii structure indicate nucleotides that have been identified as conserved in eukaryotic RNase P RNAs (Ref. 26; see text). Arced lines and bores indicate sequences that can participate in pseudoknot interactions. Lower panel, potential structures resulting from pseudoknot formation. Helix designations are those described by Forster and Altman (27). Nucleotides indicated by circles and arrows are as indicated above; boxed nucleotides are sequences that are conserved in several eukaryotic RNase P RNAs (Ref. 26; see text).

RNase P RNA from an Archaebacterium 5693 1 2 3 4 5

tRNA+3'- U

5' leader -

FIG. 6. Heterologous reconstitution of the H. volcanii RNase P RNA activity. Lane 1, pre-tRNA'"' only; lane 2, pre- tRNA""' plus H . uolcanii RNase P holoenzyme (1 rl of 100,000 X g fraction); lane 3, pre-tRNA'"' plus B. subtilis RNase P protein (13.4 nM); lane 4, pre-tRNA'"' plus H. uolcanii RNase P RNA (108 nM); lane 5, pre-tRNA'"', B. subtilis RNase P protein (26.8 nM), and H. uolcanii RNase P RNA (108 nM). Each reaction contained approximately 2.5 ng (7,000 cpm) of pre-tRNA'"'. Reaction products are indicated.

tion in these cells is greater than 2 M (34). Ionic repulsion should be quite low under these conditions. It is possible that the halophilic protein functions to induce or stabilize a catalytically competent conformation of the RNA, and the lack of activity in the absence of protein reflects the inability of the RNA to assume this conformation. In the reconstitution assay, the interaction with the B. subtilis protein may partially overcome this barrier. In addition to functioning as a charge shield, the eubacterial RNase P protein may also play some role in stabilizing the active conformation of the RNA. In the case of the E. coli RNase P RNA, many mutations that decrease or block activity in RNase P RNA-only reactions are not apparent when both RNA and protein are present (35). In these cases, the protein must overcome or correct some structural defect in the RNA. The lack of activity of the halophilic RNA in the absence of protein may also be the result of improper solution conditions or effects of flanking sequences on the RNA structure.

The gene that encodes the RNase P RNA has many of the characteristics of archaebacterial genes. In the 5' leader region of the gene are four sequences that are similar to the consen- sus archaebacterial promoter sequences (36-38): Box A, TTTAT/AATA, and Box B, ATGC, the transcription start site. Primer extension and S1 mapping studies suggest that the transcript for this gene begins at one of the 2 G residues present in the first promoter element (Figs. 3 and 4); neither analysis indicated starts in the other Box B regions. It is possible that the other Box A elements function as RNA polymerase-binding sites, priming the gene for transcription. Multiple promoters have been noted for the genes of other stable RNAs in the archaebacteria (39, 40). Alternatively, transcripts may originate from these sites followed by rapid RNA processing. Related to this, we noted the potential for the formation of a short hairpin immediately ahead of the start of the RNA (nucleotides 260-266 pairing with 276-282; see Fig. 3). Formation of this hairpin would place the proposed 5' terminus of the RNA at the base of the hairpin, possibly acting as a processing queue.

We have provided evidence for a catalytic RNA component in the halobacterial RNase P. However, this observation may not be representative of the entire archaebacterial kingdom. The RNase P activity of S. solfaturiczu contains an RNA component' but appears to differ from the halophilic activity

T. LaGrandeur, S. Darr, and N. Pace, personal communication.

in that it has a much higher protein:RNA ratio (17). We have noted in the purification of the RNase P enzyme from Ther- mophma acidophilum, a related archaebacterium, that the RNA associated with this activity is approximately 340 nucleotides. This is significantly smaller than the H. uolcanii RNase P RNA? Thus, the archaebacteria as a group may contain a variety of RNase P enzyme complexes. This apparent diversity, coupled with the demands of their unusual environments (high salt and high temperatures) on the structure of RNA and protein, make these organisms an interesting system for the study of RNase P enzymes.

Acknowledgments-We thank Drs. James Brown and Norman Pace for helpful discussions and Bernadette Pace for providing the B. subtilis RNase P protein and directions for heterologous reconstitution reactions. We also thank Dr. Ford Doolittle for providing H. uolcanii cosmids.

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RNase P RNA from an Archaebacterium 5695

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The RNA Component of RNase P from the Archaebacterium