Article No. mb981797
J. Mol. Biol. (1998) 279, 773±793
Derivation of the Three-dimensional Architecture of
Bacterial Ribonuclease P RNAs from Comparative
Sequence Analysis
Christian Massire, Luc Jaeger and Eric Westhof*
Institut de Biologie MoleÂculaire
et Cellulaire du CNRS
UPR 9002, 15 rue Descartes
67084 Strasbourg, France
The secondary structure of bacterial RNase P RNA, a ribozyme responsible for the maturation of the 50 end of tRNAs, is well established on the
basis of sequence comparison analysis. RNase P RNA secondary structures fall into two types, A and B, which share a common core formed
by the assembly of two main folding domains, but differ in their peripheral elements.
A revised alignment of 137 available sequences reveals new covariations
allowing for the re®nement of both types of secondary structures. Phylogenetic evidence is thus provided for the extension of stems P11, P14,
P19, P10.1 and P15.1 through further canonical base-pairs or GA . . . GA
mismatches. These re®nements led in turn to a new organization of the
catalytic core, with coaxial stackings of helices P2 and P19 as well as P1
and P4. New inter-domain tertiary interactions involve loop L9 and helix
P1 and loop L8 with helix P4. These features were incorporated into
atomic-scale 3D models of RNase P RNA for representatives of each
structural type, namely Escherichia coli and Bacillus subtilis. In each model,
the juxtaposition of the core helices creates a cradle onto which the pretRNA substrate binds with most evolutionarily conserved residues converging towards the cleavage site. The inner cores of both types are
stabilized similarly, albeit by different peripheral elements, emphasizing
the modular and hierarchical organisation of the architecture of RNase P
RNAs. Similarities are thus apparent between the type A modules, P16/
P17/P6 and P13/P14, and their type B analogs, P5.1/P15.1 and P10.1/
P10.1a, respectively. Other noteworthy features of these models include
compactness and good agreement with published crosslinking data.
# 1998 Academic Press Limited
*Corresponding author
Keywords: ribonuclease P RNA; ribozyme; RNA folding; RNA-RNA
interactions; RNA modelling
Introduction
Ribonuclease P (RNase P) is the endoribonuclease responsible for removing the leader
sequence from the tRNA precursors during the
maturation of the 50 end of tRNAs (for reviews, see
Altman et al., 1993; Pace & Brown, 1995). RNase P
is a ribonucleoprotein whose catalytic function, at
least in bacteria, is carried by its RNA component
(RNase P RNA) rather than by the protein
(Guerrier-Takada et al., 1983). Another remarkable
feature of RNase P RNA is its ability to recognize
the tertiary structure of its pre-tRNA substrates
(Kahle et al., 1990). The secondary structure of bacAbbreviations used: Eco, Escherichia coli; Bsu, Bacillus
subtilis; pre-tRNA, precursor tRNA.
0022±2836/98/240773±21 $25.00/0
terial RNase P RNA was inferred from a ®rst comparative analysis of sequences (James et al., 1988)
and re®ned as further sequences became available
(Haas et al., 1991; Brown et al., 1993; Haas et al.,
1994). An unexpected result was the recognition of
two main structural types, namely type A and type
B, which nevertheless share a common core (Haas
et al., 1996b). Type A is the ancestral form (see the
Escherichia coli sequence in Figure 1(a)), whereas
type B has later emerged within the Gram-positive
lineage (see the Bacillus subtilis sequence in
Figure 1(b)). Previous work on group I introns has
shown that large RNAs are likely to be organized
in self-folded structural domains (Murphy & Cech,
1993; Doudna & Cech, 1995). Such domains are
also visible in RNase P RNA, which is primarily
organized in two distinct domains (Pan, 1995;
# 1998 Academic Press Limited
774
Modelling of the 3D Structure of RNase P RNA
Figure 1 (legend opposite)
775
Modelling of the 3D Structure of RNase P RNA
Loria & Pan, 1996). Domain I, involved in the recognition of the T-loop of pre-tRNAs (Pan et al.,
1995; Loria & Pan, 1997), encompasses the ``upper
part'' of RNase P RNA, i.e. stems P7 to P12, the
large internal loop L11/12, as well as stems P13/
P14 (in type A) or P10.1 (in type B; see Figure 1).
Domain II includes the ``bottom'' half of the molecule, and comprises most nucleotides thought to
be involved in the formation of the catalytic site,
i.e. stems P1 to P5 and their ¯anking strands
(Frank et al., 1996; Pan & Jakacka, 1996; see
Figure 1). Domain II includes as well stems P15,
P19, and extensions P16/P17/P6 and P18 (in type
A) or P5.1, P15.1/P15.2 (in type B). In spite of the
early recognition of both structural types, most
attention has been focused on the type A sequence
from E. coli, for which two structural models for
the RNase P RNA-pre-tRNA complex were independently proposed (Harris et al., 1994; Westhof &
Altman, 1994). The Harris & Pace model was primarily designed as a frame for inter- and intramolecular crosslinking data (Burgin & Pace, 1990;
Nolan et al., 1993; Oh & Pace, 1994), whereas the
Westhof & Altman model was based on alternative
cross-linking data (Guerrier-Takada et al., 1989)
and chemical modi®cation data (Lumelsky &
Altman, 1988; Shiraishi & Shimura, 1988; Kirsebom
& Altman, 1989; Knap et al., 1990; Talbot &
Altman, 1994b; Westhof et al., 1996b). They share
nonetheless some common details, such as the
stacking of P1, P2 and P3, as well as an interaction
between the L15/16 internal loop and the RCCA
sequence at the 30 end of pre-tRNA (Kirsebom &
SvaÈrd, 1994; LaGrandeur et al., 1994). However,
these working models remain organised in a radically different way, re¯ecting incompatibilities
between some of the experimental data. The number of available sequences has dramatically
increased within the last two years, bringing phylogenetic evidence for long-range interaction
between L14 and P8, L18 and P8 (Brown et al.,
1996), as well as between L9 and P1 (Massire et al.,
1997). None of these contacts was actually predicted by the 3D working models, but the Harris &
Pace model has been recently re®ned to accommodate them (Harris et al., 1997). Our previous
model, however, could not incorporate these interactions without breaking the proposed catalytic
site. This fact led us to reconsider the entire modelling process on the primary basis of an exhaustive
phylogenetic analysis of the 137 bacterial
sequences available from the ribonuclease P database (Brown, 1997) and from genome sequencing
projects (see Methodology). In the present study,
we propose 3D models for the two main structural
types of RNase P RNA. Although they display
different peripheral elements, they share a similar
core centred around two inter-domain tertiary
interactions involving loops L8 and L9. The proposed models should be viewed as ``consensus''
3D structures that aim at emphasizing the folding
architecture and relationships between the folding
elements rather than precise atomic details.
Results
Type A and type B bacterial RNase P RNAs
have both been shown to be formed of two independent folding domains that can self-assemble
into a ®nal active structure (Loria & Pan, 1996).
The assembly of RNase P RNAs structural
elements is thus fully compatible with a hierarchical folding process where prefolded RNA modules
are used to create larger domains capable, once
assembled, to associate into the ®nal structure. The
strategy we have used for modelling the threedimensional architectures of type A and type B
RNase P ribozymes re¯ects this modular view of
RNA structures. In a ®rst step, we began by modelling domain I and domain II separately. Once
built, it became possible to orient and adjust the
structure of the two domains with respect to their
interdomain long-range interactions and tertiary
contacts with the pre-tRNA substrate. The resulting three-dimensional models for the architecture
of type A and type B RNase P ribozymes were
®nally re®ned with respect to the available published cross-linking data.
Modelling of domain I
The P10/P11 stack
All bacterial RNase P RNA sequences present a
wide internal loop L11/12 linking stems P11, P12,
P13 and P14 (James et al., 1988; and see Figure 1).
This loop contains two conserved regions, 130ACAGNRA-136 and 177-GUGNAA-182 (Chen &
Pace, 1997) that are found also in RNase P RNA
sequences from eukarya (PagaÂn-Ramos et al., 1996)
Figure 1. Classical representation of the 2D structure of (a) the E. coli RNase P RNA and of (b) the B. subtilis RNase P
RNA. (Haas et al., 1994). Strands involved in the formation of the P4 and P6 pseudoknots are connected by square
brackets. Extensions of stems P11, P10.1a, P15.1 and P19 are highlighted by continuous lines (see the text). Alternative
representations for the 2D structures of (c) the E. coli RNase P RNA and of (d) the B. subtilis RNase P RNA. The
alternative representations constitute an attempt at rendering the respective spatial arrangement of the helical stems
and stacks in the 3D models. Thick lines link nucleotides adjacent in the sequence with arrows indicating the 50 to 30
polarity. Nucleotides invariant among bacteria are bold. Nucleotides involved in tertiary interactions are enclosed in
boxes and linked by thin, dotted lines. Known non-canonical pairings are indicated by open circles. For each 2D diagram, the thick dotted line between P5 and P7 indicates the separation between the two main structural domains
(Loria & Pan, 1996). Stems are colour-coded according to the helical stacks proposed in the present 3D models (see
Figure 2).
776
Modelling of the 3D Structure of RNase P RNA
Table 1. Covariations between bases 128 and 230 (136
sequences)
Table 3. Covariations between bases 203 and 226 (110
sequences possessing P13/P14)
Base 128
Base 230
A
A
C
G
U
C
Base 203
G
5
126
U
Base 226
5
A
C
G
U
A
2
C
3
G
101
U
4
Data are collected with the program COSEQ (unpublished).
and archaea (Haas et al., 1996a). In spite of this
homology, the pairing that closes loop L11/12 varies between kingdoms. In bacteria, L11/12 is
closed by a composite helical structure formed of
the 2-bp stems P10 and P11 (TallsjoÈ et al., 1993;
Haas et al., 1994) and the lone base-pair 128:230
(see Table 1 and Mattson et al., 1994). In eukarya,
stems P10 and P11 are merged within a single
stem of at least 6 bp (Tranguch & Engelke, 1993),
whereas a continuous 8 or 9 bp stem P10/P11 may
form in the archaea Methanococcus jannaschii and
Archaeglobus fulgidus. This suggests that, in bacterial RNase P RNA, P10, P11, and the 128:230
interaction could also be part of a long irregular
helix. Nucleotides adjacent to the 128:230 interaction have been proposed to form additional
base-pairs (Mattson et al., 1994; Westhof & Altman,
1994). Indeed, the base-pair 129:229 shows transversions from C:G to A:G and A:U (but no C:U is
observed, see Table 2). The putative base-pair
127:231 is much more conserved, however. When
the two adjacent bulging nucleotides A232 and
A233 are present (in all bacteria but bacteriodes),
this base-pair exhibits a strong bias towards G:U
and G:C pairs (then mostly in thermophilic
species). Interestingly, in the 113 RNase P RNA
sequences displaying the bulging adenine bases
A173 and A174 at the base of stem P12, a similar
pattern of variation is observed for the adjacent
144:172 base-pair. We suggest, therefore, that the
127-G . . . 231-YAA motif should be viewed as a
part of the 7 bp stem P10/P11, in the same way
the 144-G . . . 172-YAA motif is incorporated into
stem P12 (see Figure 1(c)). A233 has been shown to
be involved in the formation of a contact with the
hydroxyl O20 of residue 62 in the pre-tRNA, which
suggests that this internal loop motif, G . . . YAA, is
particularly well suited for tertiary interactions. In
fact, in our A type model, 144-G . . . 172-YAA is
proposed to interact with loop L13 (see below).
Table 2. Covariations between bases 129 and 229 (135
sequences, both bases are deleted in the Hbc. mobilis
sequence)
The P13/P14 stack
Type B and most b-purple bacteria sequences do
not possess hairpins P13 and P14, which, otherwise, always appear simultaneously in loop
L11/12 (see Figure 1(a) and (b)). This suggests
that, in type A RNase P RNA, these stems are
coaxially stacked, so that their 50 and 30 dangling
strands are brought close to each other (see
Figure 1(c)). Interestingly, the secondary structure
of P14 can be re®ned in order to leave an identical
number of unpaired nucleotides in the L11/12
loop of type A and B RNase P RNAs (see
Figure 1(c) and (d)), in agreement with the high
level of sequence conservation of L11/12. Indeed,
in all type A RNase P RNAs, stem P14 can be
extended by joining nucleotide(s) in J13/14 to adjacent nucleotide(s) in J14/11, leaving in most
sequences the conserved G225 bulging (see Tables 3
and 4). Phylogenetic evidence for this extension is
mainly provided by ¯avobacteria sequences, where
the bulge in P14 is absent and where all WatsonCrick combinations are encountered for the basepairs extending P14. Thus, the length of P14 is
more constant than it was previously proposed (9
or 10 bp long, instead of 7 to 9 bp) in agreement
with the tertiary interaction involving L14 and P8
(Brown et al., 1996).
The stem P10.1
Among the 18 type B sequences known to date,
15 possess a P10.1 extension between P10 and P11
(see Figure 1 and 3). Phylogenetic evidence for a
tertiary interaction between P10.1a, the distal part
of P10.1, and L12, the tetraloop capping stem P12,
was previously reported (Tanner & Cech, 1995). In
most type B sequences, this interaction involves
the recognition of a conserved GAAA tetraloop in
L12 by an 11 nt GAAA receptor of consensus Bsu
145-CCUAAG . . . 159-UAUGG in P10.1a (Costa &
Michel, 1995). This interaction is expected to be
Table 4. Covariations between bases 203.0 (5' to base
203) and 227 in the 51 sequences having base 203.0
Base 129
Base 229
A
C
G
U
A
28
13
C
94
Base 203.0
G
U
Base 227
A
C
G
U
A
44
C
1
G
5
U
1
777
Modelling of the 3D Structure of RNase P RNA
similar to the P5b-P6a interaction seen in the crystallographic structure of the P4-P6 domain of the
Tetrahymena group I intron (Cate et al., 1996a).
Although the Clostridium innocuum sequence contains a GAAA loop L12, a particular loop receptor
rich in G:U pairs has been suggested for this
sequence in lieu of the expected 11 nt receptor
motif (Haas et al., 1996b). This receptor can be
rearranged as a variant of the 11 nt motif (see
Figure 3). Indeed, the resulting motif CCUGUG . . . UAUGG, with a GU platform instead of the
canonical AA platform, has actually been found by
in vitro selection to interact speci®cally with GAAA
tetraloops (see sequence C7.10 in Figure 5B of
Costa & Michel, 1997). The L12-P10.1a interaction
is also feasible in the Brevibacillus brevis sequence,
this time through a GYRA loop-helix interaction
(Michel & Westhof, 1990; Jaeger et al., 1994).
Most non-mycoplasma sequences possess an
internal loop L10.1 of consensus Bsu 139RAAA . . . 166-RAGUA and can, therefore, be
aligned with con®dence (see Figure 3). Strikingly,
this internal loop motif is similar to the loop E of
eukaryotic 5 S RNA (Shen et al., 1995), which is
characterized by a well-de®ned structure involving
the stacking of non-canonical base-pairs. By analogy, we propose the formation of the following
non-canonical pairs within L10.1 (see also Figure 3):
R139:A170 (sheared), A140:U169 (Hoogsteen),
A141:A167 (Hoogsteen) and A142:R166 (sheared).
As a result, the P10.1 extension should appear as a
straight, irregular helix that keeps constant the distance between the base of P10.1 and the 11 nt
GAAA receptor (see Figures 1(d), 2(b) and (d)).
This is emphasized by the observation than the
loop E motif L10.1 can be slide one base-pair, since
it does not affect the overall length of P10.1/
P10.1a. Even the Bacillus megaterium sequence
which possesses a longer AAAAACC . . . CGAGUA
internal loop (extra nucleotides are underlined) but
lacks two base-pairs in P10.1a can potentially have
a similar overall length for its P10.1/P10.1a extension. The formation of an AC platform, followed
by a C:C pair can indeed substitute for the two
missing base-pairs (Figure 3; and see Cate et al.,
1996b). This latter motif has actually been recognized as an alternative for the AA platform and
the G:U pair within the GAAA receptor (see
sequence C7.16 in Figure 5B of Costa & Michel,
1997). The overall stacking of the P10.1 extension is
moreover con®rmed by the remaining type B
sequences: the Acholeplasma laidlawii sequence lacks
the L10.1 internal loop and possesses instead a continuous P10.1 stem and, in the C. innocuum
sequence, the three purine:purine pairs that constitute L10.1 are likely to stack on each other. Interestingly, when the L12-P10.1a interaction is
present, the overall length of P10.1/P10.1a seems
to correlate with the length of P12: indeed, the
three mycoplasma sequences M. capricolum, M.
pneumoniae and M. genitalium display a simultaneous shortening of stem P12 and of the L10.1
internal loop, while retaining the L12-P10.1a interaction.
Analogy between P13/P14 and P10.1a/P10.1
It was previously suggested that P10.1a/P10.1
could be a structural equivalent for the P13/P14
stems (Loria & Pan, 1996). The fact that the RNase
P protein component from E. coli can bind either to
a type A or to a type B RNase P RNA could plead
for a high level of similarity between tertiary structures of both types of RNase P RNA (GuerrierTakada et al., 1983). In type A sequences, the loophelix interaction L14-P8 brings P14 close to the 3 nt
bulge between P10 and P11, i.e. close to the insertion site of P10.1 in type B sequences (see Figures 1
and 2(c) and (d)). The type B stem P10.1a/P10.1
could therefore take the place of the type A stems
P13/P14 if one assumes a tetraloop conformation
for the GAAA fragment that links P10.1 and P11 in
most type B sequences (see Figure 1(c) and (d)). By
considering the analogy between P13/P14 and
P10.1a/P10.1, one could expect that a tertiary interaction, speci®c to type A sequences, replaces the
type B interaction L12-P10.1a. Actually, P13, which
is always 6 bp long and is capped by a loop of
consensus GUAAGAG, could be involved in a tertiary contact with the 144-G . . . 172-YAA motif at
the base of stem P12. Indeed, Brown et al. (1996)
have already noticed that the stem ± loop P13/L13
and the 144-G . . . 172-YAA motif always appear
simultaneously, both features having been deleted
in type B and b-purple bacteria sequences. Interestingly, the mitochondrial RNase P RNA from
Reclinomonas americana lacks both the L13 loop and
the P12 bulge (Lang et al., 1997). Since mitochondrial sequences have been derived from the a lineage of purple bacteria, the Recl. americana sequence
provides another instance of the simultaneous deletion of the P12 bulge and the L13 loop, providing
further evidence for their implication in a common
interaction. However, due to the lack of experimental evidence for speci®c contacts between L13
and P12, we did not attempt to model this
hypothetical interaction in detail.
Conformation of the P7/P8/P9/P10
cruciform junction
The juxtaposition of stems P7 to P10 creates a
cruciform in the RNase P RNA structure (Haas
et al., 1994). In such a four-way junction, stems are
expected to be stacked by pairs, leaving only two
theoretically feasible conformations: a P7/P8 and a
P9/P10 stack or, alternatively, a P7/P10 and a P8/
P9 stack. We constructed the cruciform junction
with both conformations. Only the latter was
found compatible with the geometry of the L14-P8
interaction and with the simultaneous extension of
stems P10/P11 and P14, which leaves at most two
nucleotides between P14 and P11 (see Figure 1(a)
and (c)). This conformation is moreover the only
one that is in agreement with the cross-link of pre-
Figure 2. Ribbon representations of the complete 3-D models of RNase P RNA. Front view of (a) the E. coli model
and of (b) the B. subtilis model. The colour code is the same as in Figure 1. Stems of domain I are red, green and
deep blue; stems of domain II are orange, purple, light blue and yellow. The invariant nucleotides of the catalytic site
are represented with large white spheres, while the conserved nucleotides from the T-loop recognition site are represented with smaller spheres. Back view of (c) the E. coli model and of (d) the B. subtilis model. A rotation of 180
along the vertical axis has been applied to each model between front and back views. The loop-helix interactions
L9-P1, L8-P4, L14-P8, L18-P18 in the E. coli model, and L12-P10.1a and L15.1-P5.1 in the B. subtilis model are indicated by wide arrowheads. The ribbon drawings were made with the DRAWNA software (Massire et al., 1994).
779
Modelling of the 3D Structure of RNase P RNA
Figure 3. Re®ned alignment of the Bsu 132 . . . 216 region (equivalent to Eco 120 . . . 176) for the 18 type B bacterial
sequences. Canonical Watson-Crick pairs are green and wobble pairs are dark blue. Hoogsteen pairs are light blue
and sheared pairs are red. AA platforms and GAAA loops are purple. Unpaired nucleotides are black. Nucleotides
invariant within these sequences are bold. For clarity, the terminal loop L10.1a and the most conserved part of
J11/12 are not displayed. Instead, the number of corresponding nucleotides is indicated between square brackets for
each sequence.
tRNA G64 with both A118 in L9 and G100 in L8
(Nolan et al., 1993).
In the present models, domain I is therefore
made of the tight assembly of the nearly coaxial
stacks P9/P8, P11/P10/P7, and alternatively P13/
P14 or P10.1a/P10.1 (see Figure 2). The position of
stem P12, capping the P11/P10/P7 stack, is uncertain due to the lack of evidence for speci®c contacts
in the large loop L11/12, but is nevertheless partially constrained by the known geometry of the
L12-P10.1a interaction in the B. subtilis model.
Modelling of domain II
The stem P19
Stem P19 appears sporadically between P2 and
the well-conserved fragment J19/4 of consensus
ACARAA. Fragment J19/4 constitutes with the 30
strand of P4 the last universally conserved region
(Chen & Pace, 1997) and is unchanged regardless
of the occurrence of P19. Stem P19 is present in
most branches of the bacterial tree, except in g-purple bacteria (other taxa provide evidence for a late
deletion of P19). We noticed that in nearly all proposed secondary structures of RNase P RNA with
P19, the distance between P2 and P19 is the same
as that between P19 and the ACARAA consensus
fragment (from zero to three nucleotides). However, the secondary structure of stem P19 can be
re®ned in order to leave no unpaired nucleotide
between P2 and P19 as well as between P19 and
the conserved part of J19/4. Table 5 summarizes
the different types of pairing that are found within
the six proximal base-pairs of P19 (see also
Figure 1(b) and (d)). Although pairs Bsu 329:368
Table 5. Comparative analysis of the pairing types within the six proximal base-pairs
of P19 (B. subtilis numbering)
Base-pair
329:368
330:367
331:366
332:365
333:364
334:363
No. of
sequences
Regular
R:R
88
86
86
83
83
79
57
65
75
75
72
69
21
17
4
3
6
4
Pairing type
A:C
Mismatch
7
3
4
7
5
5
6
Pairs are grouped in the following way. Regular, C:G, G:C, A:U, U:A, G:U and U:G; R:R: G:A, A:G
and A:A; mismatch, C:C, U:U, C:U, U:C, C:A and G:G.
780
and 330:367, located at the base of P19, tend to
form more non-classical base-pairs than 331:366
to 334:363, they nevertheless favour regular
Watson-Crick base-pairs, or alternative A:A, G:A,
A:G or A:C pairs rather than other mismatches.
Purine:purine pairs have been observed in crystallographic structures extending regular helical
stems, e.g. base-pair G26:A44 in tRNAAsp (Westhof
et al., 1985) and in the P4-P6 domain of group I
introns (Cate et al., 1996a). Similarly, A:C pairs are
observed at the edge of RNA helices; for example,
an A:C pair is found at the edge of the anticodon
stem of tRNAAsp (Westhof et al., 1985). Therefore,
the presence of purine:purine or A:C pairs at the
base of P19 is fully compatible with the extension
of the helical stack. As a consequence, stem P19
appears to be adjacent to P2 in bacteria, as already
noticed in eukarya (Tranguch & Engelke, 1993).
Main helical stacks in domain II
Since the major driving force for RNA folding is
base stacking, a single stem is likely to be inserted
in a given structure at the top of an already existing helical stack. Thus, the fact that the single stem
P19 is adjacent to P2 suggests a P19/P2 stack. The
sequences that lack P19 possess instead a 4 nt linker (R19) between P2 and the ACARAA fragment
J19/4. Fragment J19/4 is highly conserved and
should therefore adopt the same conformation in
all bacterial RNase P RNA sequences. Consequently, the 30 ends of P19 and R19 should also
adopt a similar conformation in both type A and
type B models. This is achieved if R19 adopts a
loop conformation, mimicking the right-handed
stack between P19 and P2 (see Figure 4). Thus, in
both 3D models, the conserved J19/4 fragment lies
Figure 4. Close-up stereoview of (a) the junction
between R19 and P4 in the E. coli model and (a)
between P19 and P4 in the B. subtilis model. The four
nucleotides forming R19, C343 to G346, are drawn as
sticks in the E. coli model.
Modelling of the 3D Structure of RNase P RNA
on the edge of the deep groove of P2 and along the
shallow groove of P4. At the other end of P2, a
single, invariant nucleotide (G19) is inserted
between P2 and P3 (see Figure 1). In the absence of
any other feasible alternative stacking, we conserved in our models the P2/P3 stack already present in previous models (Harris et al., 1994;
Westhof & Altman, 1994). Therefore, the assembly
of P19, P2 and P3 forms the ®rst continuous helical
stack in domain II (see Figure 1 and 2).The choice
of the P19/P2 stack in our models prevents the
once proposed stacking of P1 on P2. Instead, we
propose to stack P1 on P4, since a single conserved
nucleotide (A361) is always inserted between the 30
strands of P4 and P1 (Haas et al., 1996b). P4 is in
turn likely to stack on P5, because their 50 strands
are always contiguous (Harris et al., 1994). Finally,
P5 is adjacent to both P7 and P5.1 in type B
sequences. However, P5 and P7 belong to different
folding domains and are not adjacent in some cyanobacteria sequences, whereas P5 and P5.1 are
always contiguous. Moreover, the fact that the
single stem P5.1 is present in type B sequences
argues for its stacking on a conserved stem, i.e. P5.
Thus, P1, P4, P5 and P5.1 constitute the second
continuous helical stack in domain II (see
Figures 1(d) and 5(b)).
The P15/P16/P17/P6 extension in type A RNase
P RNA
In our models, stem P15 is the only conserved
stem from the domain II that is not part of the
main helical stacks. P15 is poorly constrained in
type B sequences because of the lack of evidence of
speci®c contacts within the conserved dangling
strands J5/15 and J15/18. In type A sequences,
however, P15 constitutes the base of the P15/P16/
P17/P6 extension, which is strongly constrained by
the formation of pseudoknot P6. The orientation of
P15 may therefore be inferred from the modelling
of the whole P15/P16/P17/P6 extension from type
A RNase P RNA. P6 is usually 4 bp long, although
its size can reach 7 bp in a-purple bacteria and cyanobacteria (Vioque, 1997). One to four nucleotides
link P5 and P6, whereas a single adenosine base
joins P6 to P7 in most cases. Therefore, P6 was
stacked below P7 in the E. coli model (see
Figures 1(c) and 5(a)). The other end of P6 is
always adjacent to the 30 strand of P17, justifying
the stacking of P6 and P17 within a type III pseudoknot (see Figure 5(a) and Westhof & Jaeger,
1992). Since the P15/P16/P17/P6 extension is constrained at both ends, it should contain a sharp
turn either between P16 and P17, or between P15
and P16 (see Figures 1 and 2). The internal loop
between P16 and P17 is extremely variable in composition or in length, and is often interrupted by
the insertion of an extra stem, either between P16
and P17 (e.g. in planctomycete or a-purple bacteria
sequences, see Brown, 1997) or between P17 and
P16 (e.g. in Chlamydiae, see Herrmann et al., 1996).
The fact that these extra stems are likely to stack
781
Modelling of the 3D Structure of RNase P RNA
1994). An NMR structure of a 31-mer RNA comprising P15, P16 and the L15/16 internal loop
(Glemarec et al., 1996) shows that the bases within
the internal loop L15/16 stack upon each other,
leaving the Watson-Crick side of G292 and G293
available for additional pairings. More recently, the
L15/16 loop and its homologue in B. subtilis, L15,
were modelled in detail on the basis of a phylogenetic analysis (Easterwood & Harvey, 1997). Our
model of this region, which does not differ signi®cantly from the latter work, presents a nearly coaxial stacking of P15 and P16, allowing the pre-tRNA
30 -RCCA sequence to interact in the minor groove
of L15/16.
Putative interaction between L5.1 and L15.1 in
type B RNase P RNA
Figure 5. Close-up stereo view of the junction (a)
between P5, P6/P17 and P7 in the E. coli model and (b)
between P5, P5.1 and P7 in the B. subtilis model.
Unpaired nucleotides A79, U80, A81, A86 and G279 in
the E. coli model and A81 to U85 in the B. subtilis model
are drawn as sticks.
on either P16 or P17 strongly suggests the presence
of a kink between P16 and P17 (see Figures 1 and
2). The L15/16 internal loop, or its homologue in
type B sequences, L15, is involved in intermolecular interactions with the pre-tRNA 30 -RCCA
sequence (LaGrandeur et al., 1994) through at least
two canonical base-pairs (Kirsebom & SvaÈrd,
Type B RNase P RNA sequences lack the P16/
P7/P6 extension that contributes to the stabilisation of type A RNase P RNA structrures via the
formation of the long-range interaction P6 (Haas
et al., 1991). Nevertheless, they are characterized by
the additional stems P5.1 and P15.1 that could
potentially form an interaction in order to stabilize
domain II (Haas et al., 1996b). The recent availibility of further type B sequences allowed us to reconsider the previous alignment of P5.1 and P15.1 (see
Figure 6). Stem P15.1, previously considered to
have 6 or 7 bp, can be extended up to 11 canonical
base-pairs in each type B sequence (see Figure 6).
This extension is made possible in some species by
replacing two contiguous base-pairs by a
GA . . . GA motif or one of its variants, or by bulging a nucleotide on the 30 strand. The L15.1 loop
Figure 6. Re®ned alignment of stems P5.1 and P15.1 for the 18 type B bacterial sequences. Canonical Watson-Crick
pairs are green, wobble pairs are blue, sheared pairs are red, nucleotides characteristic to the lone Acholeplasma laidlawii sequence are purple. Unpaired nucleotides are black. Nucleotides that are invariant within type B sequences (but
in Acp. laidlawii) are bold. Square brackets stand for fragments of sequence not shown.
782
is then reduced to the consensus RAAN(3-5)AA. It
is striking that the presence of such a loop in L15.1
appears to correlate with the presence of a highly
conserved stem P5.1 of 6 bp long and capped by
an UGNRAU loop (see Figure 6). Indeed, in the
Acp. laidlawii sequence, where L15.1 is reduced to
the well-known GCAA tetraloop (Heus & Pardi,
1991), stem P5.1 contains an extra C:G base-pair
and is not capped by a UGNRAU loop. Furthermore, at a ®ner scale, the sequences of M. genitalium and M. pneumoniae may also be distinguished
from the other in both the L5.1 and L15.1 loops
(see Figure 6). In these sequences, the L5.1 loop follows the consensus UGGAAY instead of UGHGAU (signi®cant differences are underlined, see
also Figure 6). This variation correlates with the
presence in L15.1 of a loop following the consensus
RAAN(2-3)AUAA observed in all L8 decaloops (see
below), instead of consensus RAAN(3-5)AA (see
Figure 6). These correlated changes suggest that
L5.1 and L15.1 are indeed interacting. The structure of the putative L5.1-L15.1 interaction was not
modelled in detail due to the lack of evidence for
any speci®c contact. Nevertheless, the fact that a
GCAA tetraloop in L15.1 correlates with the presence of two G:C base-pairs in P5.1 pleads for a
classical GCAA loop-helix inteaction between
L15.1 and P5.1 in the RNase P RNA from Acp.
laidlawii, in place of the more complex interaction
in the other type B sequences. Since P5.1 and P15.1
are expected to be located at the same place within
all type B structures regardless of the actual nature
of the P5.1-P15.1 interaction, the permutation of
P5.1-P15.1 modules between different type B structures should preserve the active conformation.
Therefore, in the B. subtilis model we have replaced
the native P5.1-P15.1 module by its counterpart
from the Acp. laidlawii sequence, where the relative
orientation of P5.1 and P15.1 is constrained by the
known geometry of a GNRA-loop interaction
(Jaeger et al., 1994; Pley et al., 1994). The imposed
stacking of P5.1 below P5 (see Figure 2(d)) thus
constrains the position of P15.1.
Homology between P18 and P15.1/P15.2
Stems P15.1 and P15.2 are always contiguous in
type B sequences (Haas et al., 1996b). Their simultaneous insertion 50 to the fragment Bsu 314AGANNNAU-321 (nucleotides 328 to 335 in E. coli,
see Figure 1) occurs in replacement of one or two
nucleotides (Haas et al., 1994, 1996a). Therefore,
P15.1 and P15.2 are likely to stack on each other
within an independently folded subdomain P15.1/
P15.2. Most type A sequences possess instead a
single stem P18 (see Figure 1(c) and (d)). In our
models, P18 and P15.2 are placed similarly and can
even be considered as two variants of the same
helical stem (see Figure 2(c) and (d)). We keep,
however, the different names to emphasize their
structural differences: P18 is always 8 bp long and
capped by a GNRA loop, whereas P15.2 does not
exhibit signi®cant conservation in either sequence
Modelling of the 3D Structure of RNase P RNA
or length. P18 is constrained in the E. coli model by
the L18-P8 interaction (Brown et al., 1996), whereas
P15.1/P15.2 in the B. subtilis model is instead constrained by the L15.1-L5.1 interaction. It is noteworthy that these interactions place similarly the
P18 and P15.1-P15.2 extensions behind P4, so that
the neighbouring Eco 328-AGA-330 fragment can
adopt the same conformation in both models,
which is expected, since this fragment is conserved
in all bacterial sequences.
Interdomain association
The fact that the two independent folding
domains, when synthesized separately, can selfassemble into a catalytically active complex (Loria
& Pan, 1996) suggests the formation of multiple
tertiary contacts between the two pre-folded
domains. The ®rst identi®ed interdomain interaction occurs between L18 and P8 (Brown et al.,
1996). However, RNase P RNA mutants with a deletion of P18 retain a weak catalytic activity, indicating that the interdomain association may occur
in the absence of the L18-P8 interaction (Haas et al.,
1994). We recently reported phylogenetic evidence
for another interdomain tertiary interaction
between L9 and P1 (Massire et al., 1997). The conservation of a GNRA loop L9 on top of the 5 bp
stem P9 is correlated with the conservation of a
G:C pair in the distal end of P1. Alternatively, L9
and the 30 end of P1 are complementary in at least
two Mycoplasma sequences, where they have therefore the ability to be involved in the formation of a
pseudoknot in lieu of the loop-helix interaction.
Nonetheless, the L9-P1 interaction is absent in onethird of the sequences, indicating that additional
interdomain contacts may still remain to be identi®ed.
Putative interaction between L8 and P4
L8 is a good candidate for such an additional
interaction. In type A RNase P RNA, P8 is indeed
the anchoring site for both L14 and L18, suggesting
that P8 itself should be tightly bound to the core.
Moreover, in all bacterial RNase P sequences, there
is a striking conservation of two adenine bases
within L8 (Eco A98 and A99 or Bsu A105 and
A106, see Figures 1 and 7(a)), pointing to their
potential role in folding or/and catalytic function
of bacterial RNase P RNA. Interestingly, we have
noticed that 11 to 12 bp separate these conserved
adenine bases from loop L9, which was suggested
to interact with P1 in the majority of RNase P RNA
sequences (Figure 1(c); and see Massire et al., 1997).
Since P8 and P9 are stacked, loops L8 and L9 are
separated by a complete helical turn and point in
the same direction. The L1-L9 interaction could
thus act as a ruler for positioning the conserved
adenine bases of L8 in front of a speci®c site of
interaction. Considering that P1 and P4 are stacked
in our current RNase P RNA models, the best candidate for this kind of interaction is P4. Two con-
Modelling of the 3D Structure of RNase P RNA
783
Figure 7. Alignment of stems P4, P5, P7 and P8 for eight bacterial sequences representative of the structural diversity
within these stems (only the ®ve proximal base-pairs of P4 are shown). Paired nucleotides are colour-coded as in
Figure 1. Unpaired nucleotides are black. Square brackets stand for fragments of sequence not shown. Invariant bases
are bold. The ®rst group of sequences contains type A sequences with a 5 bp stem P8 capped by a pentaloop L8 conserving Eco A98 and A99. The second group gathers together the type A sequence from Chlorobium limicola (which
possesses a long P8 interrupted after 6 bp by two G:A pairs) and type B sequences (with a 6 bp stem P8 and a variable loop conserving Bsu A105 and A106). Within each group, the insertion of one base-pair in P5 compensates for
the insertion of one nucleotide in P7 (blue positions), in agreement with an antiparallel orientation of P5 and P7.
When the second group is compared to the ®rst, the insertion of one base-pair in P5 compensates for the deletion of
one nucleotide in P8 (positions marked with an asterisk), in agreement with a parallel orientation of P5 and P8. (b) A
drawing of the proposed orientation of stems P5, P7 and P8. Stems are represented by vertical arrows of proportional
length for the same sequences as in (a). The distance between the conserved bases Eco G356-G357 in P4 and A98-A99
in L8 remains the same when P5 and P7, as well as P7 and P8, are in antiparallel orientations.
served G:C pairs within core element P4 are,
indeed, located at a distance of one helical turn
from the two base-pairs supposed to interact with
L9 (see Figure 1(c)). Therefore, whereas L9 interacts
with the shallow groove side of 3:371 and 4:370
base-pairs of P1 in our type-A model, the two adenine bases of L8 interact with the shallow groove
side of the conserved Eco C71:G356 and C70:G357
base-pairs of P4 (see Figure 1(c) and (d)). Although
this putative tertiary interaction is not corroborated
by a direct covariation, its formation is fully compatible with both type A and type B models, and
can help in understanding the subtle variations
observed for the length of the stems P5, P7 and P8,
which are the structural elements separating P4
from L8. Most type A and type B sequences are
characterized by a different number of nucleotides
separating the base of stem P8 and the two conserved adenine bases in L8 (see Figure 7(a)). Interestingly, this length variation within stem ± loop
P8/L8 correlates with the length variations of
stems P5 and P7 (see Figure 7(a)). If one assumes
an antiparallel orientation between P5 and P7 as
well as between P7 and P8, the anchoring point of
P5 to P4 is then located at a conserved distance
from the two conserved adenine bases of L8, in
both type A and type B RNase P structures (see
Figure 7(b)). Therefore, the positioning of the conserved adenine bases of L8 in front of the fourth
and ®fth C:G base-pairs of P4 is possible in both
784
Modelling of the 3D Structure of RNase P RNA
type A and type B three-dimensional models (see
Figure 1(c) and (d)) and can justify the concerted
changes of length observed among RNase P
sequences within P5, P7 and P8. Several experimental data support the existence of a tertiary
contact between L8 and P4. Indeed, protections
towards the Fe(II)-EDTA hydroxyl radical of both
L8 and P4 are lost when domains I and II are constructed and folded separately (Loria & Pan, 1996).
Moreover, recent cross-linking data have shown
the proximity of L8 and P4 in the catalytically
active RNase P RNA (Harris et al., 1997). With this
interaction, the P8/P9 stack is anchored to the
catalytic domain at both ends through loop-helix
interactions (see the green arrows in Figure 2(c)),
providing a rigid frame for the L14-P8 and L18-P8
interactions (see the blue arrows in Figure 2(c)).
The overall three-dimensional architecture
According to our three-dimensional models, the
architecture of bacterial RNase P RNAs can be
seen as the assembly of helical stacks packed
together through the formation of long-range tertiary interactions (see Figures 1 and 2). Several
intradomain long-range interactions participate in
the independent folding and stabilization of
domains I and II. So far, no less than three longrange interactions have been found to constrain the
folding of domain I: interactions L14-P8 and L13P12 in type A RNase P RNA, and L12-P10.1a in
type B RNase P RNA. Domain II from type A
RNase P RNA is stabilized by the long-range interaction P6, whereas loop-loop interaction L5.1-L15.1
represents the P6 natural counterpart in type B
RNase P RNA. Interdomain tertiary interactions
like L18-P8, L9-P1 and L8-P4 are then favouring
the association of domains I and II into the ®nal
active structure. The core from both models presents a side-by-side assembly of the four main helical stacks: P11/P10/P7, P9/P8, P1/P4/P5 and
(P19)/P2/P3 (see Figures 1 and 2). This assembly
is smoothly bent, with an internal and an external
side (see Figure 8(a)). The internal side is concave
and acts as a cradle for the pre-tRNA substrate. It
contains also most of the universally conserved
nucleotides from P4, J5/15, J18/2, J19/4 that are
thought to form the catalytic site (see Figure 2).
Contacts with the pre-tRNA substrate are clustered
in three regions. (i) The 30 -terminal RCCA sequence
binds to the internal loop L15/16 in the E. coli
model or to the L15 loop in the B. subtilis model
(see Figure 8(b)). (ii) The catalytic site, in the neighbourhood of the 50 termini of the tRNA, comprises
J5/15, the 50 strand of P4, J18/2 as well as the conserved A351 and 352 from J19/4. (iii) The P11 stem
and the A118 from P9 are part of the recognition
site for the T-loop of the substrate (Pan et al., 1995;
Loria & Pan, 1997). The external side of the threedimensional architecture possesses, in contrast, the
most variable extensions that maintain the active
conformation of the catalytic site. On this external
side can be found the anchoring sites for the L14
Figure 8. (a) Top view of the CPK model of the B. subtilis
RNase P RNA-tRNA complex, viewed down the axis of
the T-stem axis of the tRNA. The T arm of the tRNA
lies in the cradle made by the assembly of the red,
green, purple and orange stacks (respectively P11/P10/
P7, P9/P8, P1/P4/P5 and P2/P3). (b) Side view of the
B. subtilis RNase P RNA-tRNA complex. A rotation of
90 along the horizontal axis is applied to the model
between both views. The upstream sequence of the
tRNA is not shown for clarity. The base-pairing between
L15 and the tRNA RCCA sequence is visible at the bottom of the Figure.
and L18 loops (see Figure 2(b) and (d)) and most
nucleotides involved in the binding of the RNase P
protein component (Talbot & Altman, 1994a).
Validation of the models
The structure of the E. coli RNase P RNA has
been investigated through crosslinking experiments (Burgin & Pace, 1990; Nolan et al., 1993;
Ê long crosslinking
Harris et al., 1994) using a 9 A
agent
(azidophenacyl
bromide)
speci®cally
attached to the 50 end of either RNase P RNA or
mature tRNA. The main crosslinks obtained in that
way are summarized in Table 6. For each attachment site, the crosslinked nucleotides are speci®ed,
785
Modelling of the 3D Structure of RNase P RNA
Table 6. Validation data for the E. coli model, according to available crosslinking data (Nolan et al., 1993; Harris et al.,
1994; Oh & Pace, 1994)
Ê)
Crosslinked nucleotides (inter-phosphate distance in A
Attachment
G
G
G
G
G
G
G
G
63
135
179
229
292
304
332
350
G1
G 53
G 64
335
112
112
127
247
247
247
247
(17.8)
(15.0)
(29.0)
(17.9)
(24.9)
(28.0)
(15.4)
(20.0)
248 (10.1)
111 (21.6)
67 (25.5)
351
118
118
129
248
248
248
248
(25.5)
(18.0)
(30.4)
(18.4)
(23.9)
(24.6)
(13.7)
(19.4)
331 (20.9)
114 (24.4)
68 (21.2)
352 (26.4)
123
137
253
295
295
300
(36.0)
(22.0)
(11.7)
(30.4)
(15.3)
(22.4)
332 (16.4)
348 (24.6)
100 (23.2)
128
138
260
296
(18.4)
(20.2)
(17.1)
(32.6)
132 (18.2)
183 (19.7)
‡2 (30.6)
185 (17.6)
‡3 (34.7)
329 (19.4)
-4 (19.9)
-5 (25.1)
333 (21.2)
349 (20.7)
118 (22.0)
247 (17.8)
248 (21.1)
0
The attachment site of 5 -azidophenacyl crosslinking agents is indicated in the ®rst column. For each attachment site, the crosslinked
nucleotides are indicated, with the mean internucleotide distance measured between P atoms. Nucleotides within the pre-tRNA subÊ ) are bold.
strate are indicated with italicized numbers. Distances greater than the chosen threshold value (30 A
with the mean inter-nucleotide distance (measured
between phosphorus atoms) indicated in parentheses. We consider that a given crosslink is compatible with our model if this distance is less than
Ê , since the localization of these crosslinks is
30 A
not known at an atomic scale. Moreover, since
most attachment sites are obtained by circular permutations of the RNA, crosslinking agents are
attached to new 50 ends, for which the backbone
itself is expected to add some extra ¯exibility. The
majority of intra- and inter-molecular crosslinks
are compatible with our models (see Table 6). Two
distinct sets of incompatible crosslinks are visible.
The ®rst set involves the G179 crosslinks, i.e.
within the region of L11/12, which is poorly constrained in our models. The high level of conservation of this internal loop suggests speci®c contacts
between its bases, which we did not attempt to
model. The second set of unsatis®ed constraints
occurs in the region of P15. Once again, the actual
3D structure of this region is likely to be more
compact than in our models and remains undetermined.
Discussion
General organization of the core of RNase
P RNAs
Type A and type B RNase P secondary structures share a common conserved core that encompasses parts of structural domains I and II:
roughly, stems P1 to P5 with P15 from domain II
and stems P7 to P11 from domain I (see Figure 9).
These core elements may be conserved because: (i)
they are part of the catalytic site (P4, J5/15, J19/4
and J18/2); (ii) they are involved in the correct recognition of the substrate (P11, L15); and (iii) they
form part of the frame that stabilizes the catalytic
core or constitutes the interdomain interface (P8,
P9). Therefore, deletion of some of the normally
conserved core elements may affect or abolish the
speci®city for pre-tRNA substrates, while still
allowing cleavage of alternative substrates
(Guerrier-Takada & Altman, 1992; Pan & Jakacka,
1996). Figure 9(c) illustrates the decomposition of
the core into two main domains, each formed by
the coaxial stacking of several helices. We do not
wish to convey the impression that Figure 9(c) represents the 2D structure of an Ur-RNase P, precursor of the two bacterial types, although we cannot
exclude the fact that it constitutes a possibility. The
common interdomain contacts involve P1 and L9
as well as P4 and L8. However, type A and type B
RNase P RNAs present their own set of peripheral,
tertiary elements that contribute to the stabilization
of the catalytic core through the formation of
speci®c tertiary interactions (see The overall threedimensional architecture in Results and Figure 9(a)
and (b)). Insertion sites of less conserved, peripheral elements occur on the core periphery. Type A
and type B RNase P RNAs are best distinguished
by the elements inserted in the single-stranded
regions between P5 and P7 and L15 (see Figure 9).
Several long-range interactions characteristic of
each type of RNase P occur between peripheral
elements (e.g. L5.1-L15.1 in type B or P6 in type A)
as well as between the periphery and the core (e.g.
L14-P8 and L18-P8 in type A). Despite the fact that
these peripheral elements are non-homologous,
they nevertheless have similar, functional purposes
by contributing to the folding and stabilization of a
common catalytic core.
Comparison with other models
The present models are somewhat similar, in
their general domain organisation, to the last
model described by Harris and Pace (later referenced as the HP model, see Harris et al., 1997).
Common features include a roughly similar orientation of the pre-tRNA substrate, an identical ¯attened conformation of the four-way junction, a
network of tertiary interactions L14-P8, L18-P8 and
L9-P1, and a C-shaped extension P15/P16/P17/P6.
Signi®cant divergences are, however, clearly visible, in particular in domain II. Whereas the P4/P5
and P1/P2/P3 stacks form a T in the HP model,
the stacks P1/P4/P5 and (P19)/P2/P3 are nearly
parallel in our models.
786
Modelling of the 3D Structure of RNase P RNA
Figure 9. Simpli®ed 2D representations of (a) a typical type A sequence and of (b) a type B sequence with their
characteristic tertiary interactions. Stems are colour-coded as in Figures 1 and 2 with conserved regions represented
with thick lines. Main insertion sites are indicated by arrowheads. (c) A 2D representation of the minimal core. Insertion sites common to both types (P12, P18 and P19) are indicated by small arrowheads. The insertion sites of peripheral elements that are characteristic of a given type are indicated by large arrowheads.
787
Modelling of the 3D Structure of RNase P RNA
More importantly, the incorporation of P19 in
our models leads to a different topology of the central pseudoknot with an alternative crossing of the
junctions J19/4 and J3/4 (see Figure 4). In Results,
we proposed that the conserved J19/4 lies on the
edge of the deep groove of P2 and along the shallow groove of P4, if one assumes a right-handed
stack between P19 and P2. The crossing between
J19/4 and the 50 strand of the P1 and P2 helices is
thus reversed compared to that of the HP model.
In order to avoid the passage of J19/4 between the
50 strand of P2 and J3/4, the crossing between the
J19/4 and J3/4 strands also must be reversed. As a
consequence, J19/4 lies on the substrate side in our
models, instead of being on the protein side as in
the HP model (compare the boxed 2D sketches in
Figure 10(a) and (b)).
This different placement of J19/4 has important
consequences for the sequence of folding of the
central pseudoknot. P2 was among the ®rst helices
to be ®rmly identi®ed by phylogenetic sequence
comparison (James et al., 1988) and has, therefore,
been naturally thought to be part of the secondary
structure. Evidence for four out of the eight basepairs of P4 was then also provided. Because of its
assumed shorter length, P4 has been implicitly considered to fold after the formation of P2. This hierarchical folding is present in the HP model: the
unfolding of its pseudoknot starts with the disruption of P4 (see Figure 10(a), right) and not by the
opening of P2, which would lead to an entanglement (see Figure 10(a), left). As new sequences provided new instances of covariations in the distal
base-pairs, P4 has been extended to its present
length of 8 bp plus a bulge (Haas et al., 1991).
However, the folding of the central pseudoknot
has not been re-analysed. The following facts
argue for an alternative folding of the pseudoknot:
(i) P4 is indeed longer than P2 (8 bp and a conserved bulge versus. 7 bp); (ii) P2 displays greater
variation in length than P4, and is even absent in
some mitochondrial sequences where P4 lacks only
one base-pair; (iii) protections towards the Fe(II)EDTA hydroxyl radical show that P2 is almost
completely exposed to solvent, whereas P4 is
deeply buried in the structure (Loria & Pan, 1996);
and (iv) experimental evidence for a slower formation of P2 has been reported (Zarrinkar et al.,
1996). These arguments would indicate that P2 is
indeed a tertiary interaction with P4 forming the
2D structure. This is noticeable in our models (see
Figure 10(b), right), where the 30 strand of P2 and
its ¯anking junctions, J18/2 and J19/4, make a
broad loop that, upon folding, creates the pairing
of P2. One can therefore expect that the unfolding
of the pseudoknot starts with the disruption of P2
and not of P4 (see Figure 10(b), left).
In domain I, the main difference between the HP
model and our models concerns the positioning of
P13 and P14. In our models, the fact that P13 and
P14 are stacked implies a rather different orientation of P13 and P12. The latter stem is partially
constrained by the maintenance of the L12-P10.1
interaction. But, since P10.1 is itself constrained
only in its lower part, small variations in the structure of the P10/P10.1/P11 junction would result in
greater changes in the position of the distal end of
P10.1. This would dramatically alter the orientation
of P12. This is the reason why the modelling of
P12 and L11/12 will probably need re®nement.
Long-range interactions within RNase P RNAs
RNA-RNA long-range interactions
The long-range interactions within RNase P
RNAs belong essentially to two broad categories:
base-pairing between two loops and the family of
GNRA loop-receptor interactions (Westhof et al.,
1996a; and see Figure 1). Interestingly, both kinds
of contacts are alternatively found between L9 and
P1, since this interaction may be realized either
through a GNRA-helix interaction (mostly in type
A sequences) or through the formation of a pseudoknot P21 in some mycoplasmas (Massire et al.,
1997). Nevertheless, GNRA loop-receptor interactions are found at four distinct locations and are
thus the most widespread long-range interactions
among RNase P RNAs.
In addition, several tertiary interactions proposed in our models involve adenine-rich terminal
loops (L8, L13, L5.1 and L15.1) distinct from the
classical GNRA tetraloop, as well as adenine-rich
internal loops, such as those found within P10/P11
and P12. Recently, Abramowitz & Pyle (1997)
reported that GNRA tetraloops may be considered
as members of a larger family of GNn . . . RA motifs
that all share the ability to form tertiary interactions through the recognition of the minor
groove of a helix. GNn . . . RA motifs may thus be
enlarged to hexa- or heptaloops, or even internal
loops if the insertion sequence has the ability to
fold itself into a regular hairpin stem ±loop structure. Such a motif is indeed found in the Deinococcus radiodurans sequence, which displays an
unusually long P9 stem interrupted by a
GA . . . GAA internal loop after 5 bp, i.e. at the
exact place of the L9 tetraloop found in most
sequences. Accordingly, the G:C pairs that constitute the putative loop receptor are seen in P1 as
expected. Other probable members of the
GNn . . . RA motif family are the L8 decaloop and
the L15.1 loop from type B RNase P RNAs, of
respective consensus GAAN3AUAA and GAAN35AA (see Figure 7). The conservation of both ends
suggests that these loops are closed by at least one
sheared G:A or A:A base-pair, thus extending the
helical stacks P8 and P15.1. The Watson-Crick side
of at least the 30 adenine base is therefore available
for further long-range interaction, as is the case
with a canonical GNRA tetraloop. Thus, whereas
adenine bases of the L8 decaloop are proposed to
interact with the shallow groove of the G:C basepairs within P4, adenine bases of L15.1 could well
be involved in a new type of loop-loop interaction
with the hexaloop UGNRAU, L5.1. The higher
788
Modelling of the 3D Structure of RNase P RNA
Figure 10. Schematic views of the unfolding (upper arrows) and folding processes (lower arrows) of domain II as a
function of the conformation of the P2-P4 pseudoknot common to RNase P RNA. (a) Topology found in previous
models (HP). (b) Topology in present models (MJW). The secondary structure schemes in the rectangles represent the
folding topology of the central P2-P4 pseudoknot and its connections to the surrounding P1, P3 and R19/P19. The
insertion of stems P5 to P18 is indicated by two dots. In (a), J19/4 is behind J3/4 and the 50 strand of P1 and P2,
while in (b) it is the reverse. Paths to the right illustrate possible unfolding pathways. In (a), ®rst P4 must open and
then P2, while the opposite occurs in (b). Paths to the left illustrate unfolding pathways leading to strand entanglements. The process of unfolding the central pseudoknot P2-P4 is thus non-commutative, in HP models ®rst P4 opens
and then P2, while in MJW models ®rst P2 opens and then P4. The non-commutativity of the two operations comes
formally from the topology of the junctions between the central pseudoknot and the connecting helices. Thus, in the
HP model, if the relative positions of P3 and domain I were interchanged, the path to the left would be possible (a).
Similarly, in MJW models, if the junctions J1/4 and J3/4 were interchanged, the path to the left (b) would be possible. However, the assumed right-handed stacking between P2 and P3, as well as between P1 and P4, leads to the
stereochemically favoured foldings shown in the boxed schemes. Further, the choice for the MJW model compared to
the HP model derives from the right-handed stack between P19/R19 and P2.
degree of conservation within the decaloop L8 is
moreover striking, and suggests speci®c pairings
between 50 and 30 ends of the loop. Internal loops
of similar sequence GAAN . . . AUAA are found
also within stem P9 of purple bacteria sequences
(Brown et al., 1996) in place of the GNRA loop L9,
providing additional evidence for the implication
of such motifs in tertiary interactions.
In type A RNase P RNAs, the natural counterpart of the L8 decaloop is a pentaloop of consensus
UAAYR, which is also proposed to interact with
the G:C base-pairs in P4. This loop has no obvious
similarity with the classical GNRA loop and seems
to be much less widespread.
The G . . . YAA internal loop motif is another
interesting motif found at two different locations
among RNase P RNAs (see Figure 1(c)). This motif
within P10/P11 was shown to recognise speci®cally the pre-tRNA backbone (Pan et al., 1995;
Loria & Pan, 1997). As proposed in our 3D models,
the same motif within P12 is potentially involved
in the recognition of the conserved heptaloop L13
of consensus GUAAGAG. It remains to be seen
whether the motif G . . . YAA is found in other
large RNA structures.
Possible compensation of a missing RNA-RNA
interaction by the protein component
The conservation of the L8-P4 interaction is of
key importance in our models, since it maintains
the association of the two main folding domains in
a cradle-like conformation. This interaction is also
expected to occur in archaea, since the archaeal
789
Modelling of the 3D Structure of RNase P RNA
consensus core is almost identical with the bacterial core (Haas et al., 1996a). However, the whole
stem ±loop P8/L8 is absent in both archaea Methanococcus jannaschii and Archaeoglobus fulgidus.
These sequences are, moreover, the only archaeal
ones where the whole P16/P17/P6 extension has
also been deleted, raising the question of a possible
link between the deletion of these structural
elements. One can nevertheless speculate that, in
both these organisms, the protein component of
RNase P has co-evolved in order to compensate for
the lack of P8 and/or P6 so as to maintain the
active conformation of the RNA. Unfortunately, no
RNase P protein from archaea has been fully identi®ed to date (Nieuwlandt et al., 1991; Darr et al.,
1992), and no analogue of bacterial (Brown & Pace,
1992) or eukaryal (Kim et al., 1997) proteins is
found in complete archaeal genomes (Bult et al.,
1996; Klenk et al., 1997; Smith et al., 1997). The
absence of a bacterial-like protein in archaeal
RNase P is particularly striking when considering
the very strong structural homology between
archaeal and bacterial RNase P RNA. In eukarya,
the secondary structure of RNase P RNA is not yet
®rmly established (Tranguch & Engelke, 1993), but
resembles the bacterial or archaeal consensus in
many points (Chen & Pace, 1997). Animal
sequences nevertheless seem to lack stems P5 and
P15, which may be linked with the emergence of a
stem-loop-stem structure within P3. Yeast
sequences may be distinguished by their unique
structure of the cruciform junction, illustrating
once again the modular use of alternative RNA
structural motifs and their possible stabilization
through either RNA-RNA or protein-RNA interactions.
Conclusion
The modelling of the three-dimensional architecture of group I introns emphasized the hierarchical
folding of large RNA molecules (Michel &
Westhof, 1990; Jaeger et al., 1996; Westhof et al.,
1996a). First, the pairings of the secondary structure join proximate regions in sequence, followed
by end-to-end stacking of contiguous helices.
Those preformed domains then associate to constitute the compact tertiary structure via interactions
between tertiary architectural motifs (for a review,
see Brion & Westhof, 1997). The ®rst recurrent 3D
motif discovered (Michel & Westhof, 1990) consists
of a GNRA tetraloop interacting with two consecutive base-pairs in the shallow groove of an RNA
helix. The primordial role played by this motif and
its variants in the hierarchical self-assembly process of large RNAs has since been established
(Jaeger et al., 1994; Murphy & Cech, 1994; Pley
et al., 1994; Costa & Michel, 1995, 1997; Cate et al.,
1996a; Costa et al., 1997). The modelling of four
complete self-splicing group I introns belonging to
four subgroups illustrated the widespread use of
two types of long-range RNA-RNA anchors
between peripheral domains: GNRA-helix receptor
and loop-loop interactions (e.g. see Lehnert et al.,
1996). The picture that emerged from that study
was that non-homologous peripheral domains,
characteristic of each subgroup, engage in either
one of those two RNA-RNA long-range anchoring
interactions and, thereby, contribute to either the
stabilization of the helical stems of the catalytic
core or the correct positioning of the helical substrate.
The present work extends the view of a modular
and hierarchical RNA self-assembly, as seen in the
architecture of group I introns, to the bacterial
RNase P ribozymes. Again, coaxial stacking of helical stems (one of which contains a pseudoknot) is
largely prevalent in the formation of subdomains
that create a cradle into which the substrate, the
amino acid arm of pre-tRNA, is bound. And,
again, the maintenance and assembly of the subdomains is performed by a recurrent and systematic
use of GNRA-helix and loop-loop anchoring contacts. Finally, again, different and non-homologous
peripheral elements promote different and
mutually exclusive long-range anchoring contacts
with identical functional purposes.
Only a fraction of group I introns self-assemble
and most of them require the help of protein cofactors for proper folding. A well-described system is
the Neurospora crassa mitochondrial tyrosyl tRNA
synthetase (CYT-18 protein), which binds to the
P4-P6 domain of introns belonging to subgroup
IA2, like the N. crassa NcLSU intron (Caprara et al.,
1996b). Those introns lack the P5abc extension,
which forms a self-folding domain (Murphy &
Cech, 1993; Cate et al., 1996a) and is situated at the
back of the substrate binding site. It has been
shown that CYT-18 binds at the same place as
P5abc (Caprara et al., 1996a). Similarly, in the present models, the P RNA forms a layer of helices
sandwiched between the protein cofactor and the
tRNA substrate. One can speculate that a fully
autonomous RNase P functioning without a protein cofactor would require the presence of
additional peripheral domains for adequate stabilization of the helical core at the back side of the
substrate recognition surface.
Methodology
Phylogenetic analysis
The initial alignment of 87 bacterial sequences from
the sixth edition of the book of P was ®rst downloaded from the ribonuclease P database (Brown, 1997).
New sequences were added and manually aligned
using SeqPup (Don Gilbert, Indiana University) as they
were directly brought to publication (J. W. Brown, personal communication; and Haas et al., 1996b;
Herrmann et al., 1996; Vioque, 1997) or derived from
genome sequencing projects (Fleischmann et al., 1995;
Fraser et al., 1995; Himmelreich et al., 1996; Tomb et al.,
1997). Although the sequencing of the complete gen-
790
Modelling of the 3D Structure of RNase P RNA
Table 7. References and access numbers of the sequences cited in this work
Organism
Reference
Acholeplasma laidlawii
Archaeglobus fulgidus
Bacillus megaterium
Bacillus subtilis
Brevibacillus brevis
Chlamydia trachomatis
Chlorobium limicola
Clostridium innocuum
Deinococcus radiodurans
Escherichia coli
Methanococcus jannaschii
Mycoplasma capricolum
Mycoplasma fermantans
Mycoplasma genitalium
Mycoplasma pneumoniae
Reclinomonas americana
Synechococcus PCC6301
Haas et al. (1996b)
Klenk et al. (1997)
James et al. (1988)
Reich et al. (1986)
James et al. (1988)
Herrmann et al. (1996)
Haas et al. (1994)
Haas et al. (1996b)
Haas et al. (1991)
Reed et al. (1982)
Bult et al. (1996)
Ushida et al. (1995)
Siegel et al. (1996)
Fraser et al. (1995)
Himmelreich et al. (1996)
Lang et al. (1997)
Banta et al. (1992)
ome of Neisseria gonorrhoeae, Streptococcus pyogenes, (Roe
et al., 1997, http://dna1.chem.uoknor.edu, Neisseria
meningditis, Vibrio cholerae (TIGR, 1997, http://www.
tigr.org/tdb/mdb/mdb.html) and Treponema pallidum
(Weinstock & Norris, 1997, http://utmmg.med.uth.
tmc.edu/treponema/tpall.html) are still under progress,
their unique RNase P RNA gene (rnpB) has been identi®ed by BLAST searches (Altschul et al., 1997). These
sequences contain the whole set of conserved nucleotides that are characteristic of bacterial RNase P RNA.
They display therefore a canonical type A secondary
structure and have been added to the alignment.
Sequences were then classi®ed by phylogeny before
being sought for ®ner alignment within the most variable stems (P3, P6, P9, P12, P16, P17, P19, P10.1) and
within single-stranded regions (L11/12, L15/16, L8,
L15.1). For clarity, references and access numbers of
sequences of particular interest are grouped in Table 7.
Throughout the text, any base-pair is denoted by a
colon. Residues involved in internal loops are separated by three dots. Other standard abbreviations are:
N, any nucleotide; R, A or G; Y, U or C; H is A, C or
U. Nucleotides in bold letters represent invariant
nucleotides.
Three-dimensional modelling
Molecular modelling was performed as described
(Michel & Westhof, 1990; Westhof, 1993) using the program MANIP (unpublished) implemented on an SGI
workstation. The 3D structures were geometrically
re®ned with the restrained least-squares program
NUCLIN-NUCLSQ (Westhof et al., 1985). Drawings were
produced by DRAWNA (Massire et al., 1994) on an SGI
workstation. Coordinates are available by anonymous
ftp (130.79.17.244).
Acknowledgements
We are grateful to Dr J. W. Brown for communicating
RNase P RNA sequences from green non-sulphur bacteria prior to publication, and more generally for his
involvement in the creation and the updating of the
RNase P database (Brown, 1997). We thank Dr S. Norris
for the identi®cation of the rnpB gene in the Treponema
Access number
U64877
AE000782
M19022
M13175
M19023
U44031
L25703
U64878
M64708
M17569
L77117
D13066
U41756
L43967
U00089
AF007261
X63566
pallidum genome. C.M. ackowledges support from the
MinisteÁre de l'eÂducation nationale, de l'enseignement
supeÂrieur, de la recherche et de l'insertion professionnelle and the Human Frontier Science Program
(CRG0291/1997-M to E.W.). E.W. thanks the ``Institut
Universitaire de France'' for support.
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Edited by J. Karn
(Received 26 November 1997; received in revised form 23 March 1998; accepted 26 March 1998)