Vol. 266, No. 9, Issue of March 25, pp. 5689-5695, 1991
Printed in 11.S. A.
THEJOURNALOF BIOLOGICAL
CHEMISTRY
(c’ 1991 by
The American Society for Biochemistryand Molecular Biology, Inc.
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 $Departmentof Microbiology, Ohio State University, Columbus, Ohio43210 and the §Department of Biology,
Indiana University, Bloomington, Indiana47405
RNase P, an endoribonuclease responsible for generating the mature 5‘ termini of tRNA precursors, is
composed of both RNA and protein. It has been demonstrated 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 ofeukaryotic 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 resembling the eubacterial RNase P RNA andincludes 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 subunitof the
Bacillus subtilis RNase P complex. Similarities among
thearchaebacterial,eubacterial,andeukaryotic
RNase P RNA sequences and structures are discussed.
is no directevidence 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 mosaicof molecular characteristics.
Some of their molecular features are eubacterial-like,
whereas
othersare eukaryotic-like. For example, they have rRNA
operons and operon structures in general similar to the eubacteria. Contrasting this, some stable RNAgenes are interrupted by introns. Othergenes, like those encoding ribosomal
proteinsandRNA
polymerases,encode proteinsthatare
eukaryotic in nature (15).
Two reports havedescribed 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 havea buoyant density
of 1.61 g/cm” in cesium sulfate (16). These data are consistent
withtheproposalthatthis
enzyme has arequired RNA
component and suggest that, like the eubacterial complex, it
Archaebacteria, like the eubacteria and eukaryotes,
possess is composed of a large RNA and a small protein. A second
report described the RNase P activity from Sulfolobus solfaa n activity that removes the 5’ leader sequences from tRNA
taricus, amember of the acid thermophilebranch of the
primary transcripts. In eubacterial and eukaryotic cells, this
archaebacteria (17). This enzyme was not sensitive tomicroreaction is carriedout by the enzyme RNase P, which is
coccal nuclease and had a buoyant density of 1.27 g/cm:‘ in
composed of both protein and RNA (for reviews, see Refs. 1- cesium sulfate. This apparent
higher protein:nucleic acid ratio
3 ) . The role of the RNA component in the eubacterial RNaseresembles the eukaryotic and organellar RNase P activities.
P complex is now clear;RNAfunctionsasthecatalytic
These studiessuggested a diversityamong the archaebacterial
component (4, 5 ) . Despite thecommon feature of RNA catal- RNase P enzymes and left unresolved the role of RNA in
ysis shared by these enzymes, sequence analysis has shown catalysis.
that there is considerable divergence between these RNAs.
In this report, we describe the isolation of a gene encoding
The RNaseP RNAs from Escherichia coli and Bacillus subtilis an RNA that copurifies with H. uolcanii RNase P activity.
exhibit only 43% sequence similarity (11).Recently, a com- The transcript from this gene can assume a folded structure
mon feature hasbeen observed that again relates thesediver- that is similar to eubacterial RNase P RNA structures and
gent RNAs.A comparison of a phylogenetically diversegroup shares manyof the highly conserved sequencesof these RNAs.
of eubacteria has shown that these RNAs share a common Under a variety of i n vitro incubation conditions, this RNA
structural core, where conserved nucleotides are localized to was incapable of catalyzing cleavage of pre-tRNAs. However,
similar structural regions (12, 13). While evidence suggests
catalytic activitywas observed when this RNA was combined
that some eukaryotic RNase P complexes require RNA, there with B. subtilis RNase P protein.
* T h i s work was supported by Office of Naval Research Grant
N00014-89-5-3077. The costs of publication of this article were defrayed 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
to theGenBank””/EMBLDataBankwith
accession number&
M6 1003.
n Associate of the Canadian Institutesfor 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.
MATERIALSANDMETHODS’
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 endof 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 anArchaebacterium
FIG. 3. Sequence of the H. uolcanii RNase P RNA gene region.
Underlined sequences represent the
RNase P RNA. Arrows indicate possible
hairpins, andpotential transcription signals (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.
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 G200 in 2 M KC1 (physiologicalsalt 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 wereprepared 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 wassubcloned and subjected
to DNA sequence analysis. The sequence of this fragment is
presented in Fig. 3.
To verify thatthis 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 RNAwas not mapped,
Northern data and localization of the 5' terminus suggest
'L
The abbreviations used are: kb, kilobase(s); bp, base pair(s).
GCGGClCG~GRRRCAGCCRGClCGGC~~~RGCGCGGCROCCClllGCG~G~~ClGG
c
840
GlTCGCCGCCGC~GTCTCGGl~lGCGCGGCllG~ClClCClCGlGGCGGlGC~C~lCGlC
CRUGCGGCGGCGTCRGRGCCRTRCGCGCCG~~ClG~G~GGUGC~CCGCC~CG~Gl~GC~G
900
CTCGCCGGClGTGCCGCGCCGGlGlCGCCGGG~~CCGRCGGG~CG~RGCG~CG~ClGCC
GRGCGGCCGRCRCGGCGCGGCCRCRGCGGCCCllGGClGCCClGClGlCGCTGClG~CGG
960
UCGTClGC~GClTCGCCGCCGRCCRetGRCTCOGCRRCCGGC~~CCG~GlCGCCCGCGGCG~CCGCG
1GCRGACGCCGRRGCGGCGGClGGlGGCTG~GCCGllGGClC~GCGGGCGCCGClGGCGC
RECCCGRCGRCTTCGCCTCCGTrGRC
TGGGGClGCTGRRGCGGRGGC~GClG
b lI
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 threeloop corestructure 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
maybe 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, respectively. This RNAwas
assayed with both the halophilic
tRNAV"' substrate
and
the CCA-containing B. subtilis
tRNAAapsubstrate 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 thepresence 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
1
2
T C C A P L .
-133
nt
-462
nt
5691
T C G AS1
RNA
435 nt-
1
3
C
e
e
-191
nt
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 thecomplement 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 Insertions into the analogous helical region separating the
in a heterologous reconstitution experiment. Incubation con- central and uppermostloops in the B. subtilis RNase P RNA
ditions were optimized for the activity
of the B. subtilis RNase also affect protein interaction (32). The halophilic RNA conP RNA plus its protein. Under these conditions, the halophilic
tains both similarhelical structures and related sequences in
RNA exhibited cleavage activity with the tRNAVa’ substrate this region. Therefore, thisregion of the halophilic RNAmay
(Fig. 6). Full recovery of the activity was not possible with play a role in protein interaction. Other mutationsthat affect
the heterologous complex. Approximately 10% of the cleavage cleavage efficiency in the eubacterial RNase P RNAs have
activity was recovered when compared to thehalophilic hol- been described. These mutations are located throughout the
oenzyme. Similar results were obtained with the B. subtilis molecule, and it hasbeen difficult to distinguish “true” catatRNAAsp substrate(Fig. 7).
lytic mutations from those that have an indirect
effect on the
activity due to changes in RNA folding (1, 2). The diversity
DISCUSSION
of structures among the eukaryotic nuclear and organellar
Using cDNAs prepared against RNAsthat copurified with RNase P RNAs makes comparisons between these and the
RNase P activity from H. volcanii, we have isolated the gene other RNAs difficult. One common structural feature that
the halobacterial, eubacterial, and
that encodes the RNA componentof this enzyme. Identifica- may be shared between
tion of this RNA as a component of the RNase P complex eukaryotic RNase P RNAs and the mitochondrial RNA processing RNase, is the formation
of a pseudoknot (13, 27).
stems from three observations. Previous studies on the physical properties and nuclease sensitivity
of this enzyme strongly Interestingly, these interactions bring together most of the
suggested that this activity had a required RNA component universally conserved sequence elements that are present in
(16). Second, sequence data indicate that this RNA can as- distant regions of the molecule. Located in thisregion of the
H. uolcanii RNase P RNA are 12 of the 19 nucleotides consume a structure similar to the core structure proposed for
P RNAs
eubacterialRNase P RNAs, and it contains many
of the served between this RNA and the eukaryotic RNase
conserved sequence elements. And finally,in vitro transcripts (26) (seeFig. 5). Sequence homology between the halobacteria
P protein and eukaryotic RNase P RNAs can be extended further if
from this gene, when combined with the RNase
sequences conserved in the pseudoknot interaction are also
from B. subtilis, exhibit catalytic activity.
A comparison of the structure and sequence features
of the included. Each eukaryotic RNase P RNA contains the conhalophilic, eubacterial, and eukaryotic RNase P RNAs indi- served sequence element 5’-GNAANNUCNGNG-3’, which
of the 3”terminal loop (27) (seeFig.
cates that the halophilic RNA is most similar to the eubac- pairs with the sequences
terial RNase P RNA. Its overall structure closely resembles 5). Five of these nucleotides are conserved in theH.uolcanii
the eubacterial core structure, and it contains 46 of the 60 RNase P RNA. If all of these nucleotides are considered, then
conserved eubacterial RNase P nucleotides. Further correla- 13 nucleotides are conserved in the RNase P RNAs from all
three kingdoms. The pseudoknot structuremodel is also contions between these RNAs with respect to catalytic centers
and protein-binding sites are tentative due to the lack
of sistent with earlier mutagenesis datafor B. subtilis RNase P
understanding of these issues in the eubacterial
enzyme. Some RNA, which indicated that active site formationinvolves the
of the molecule (33).
structural regions and specific nucleotides of the RNase P interaction between distant portions
The inability of the halobacterial RNase P RNA to act
RNA, which have been ascribed to a particular function, have
catalytically in the absence
of protein is puzzling. In the
correlates in the halophilic RNA. For example, the protein
eubacterial system, the protein component appears to act as
components of E. coli and B. subtilis RNase P RNAs are
thought to interact with sequences in the uppermost
loop and a cofactor that shields theionic repulsion forces between the
substrate and catalytic RNAs (28,29). Its presence affects the
the helical region that separates this loop from the central
loop (see Fig. 5). In E. coli, the C5 protein protects regions V,,, of the reaction, but not the binding of substrate. It is
82-96 and 170-185 (2, 30). A point mutation in this region, unlikely that the protein component
of the halophilic RNase
(31). P plays thisrole since the internal monovalent ion concentraGS9 to A”’, leads to a defectinproteinassociation
5692
RNase P RNA from an Archebacterium
c 5
IV
. ..
160
IV
.
IV
HeLa
Hf. volcanii
E . GQU
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
1
tRNA+3'-
5' leader
2
3
4
5
U
-
FIG. 6. Heterologous reconstitution of the H. volcanii
RNase P RNA activity. Lane 1, pre-tRNA'"' only; lane 2, pretRNA""' 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 (108nM). Each reaction contained approximately 2.5 ng (7,000cpm) ofpre-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. subtilisprotein may partially
overcome this barrier. In addition to functioning as a charge
shield, the eubacterial RNase P protein may also play some
role instabilizing 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 onthe 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 sequencesthat are similar to theconsensus 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 and4); neither
analysis indicated starts in the other Box B regions. It is
possible that the other Box A elements function as RNA
polymerase-bindingsites, priming the gene for transcription.
Multiple promoters have been noted for the genes of other
stable RNAsin 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.
5693
in that ithas amuch higher protein:RNA ratio (17). We have
noted in the purification of the RNase P enzyme from Thermophma acidophilum, a related archaebacterium, that the
RNA associated with this activity is approximately 340 nucleotides. This is significantly smaller than theH.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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6.6
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--
48
-r)
-2.6
2.3
2.0-
w
*
0.56
5'leader
-1.0
-
-
*
n *
.