FEMS Microbiology Reviews 23 (1999) 391^406
Ribonuclease P:
the diversity of a ubiquitous RNA processing enzyme
Astrid Scho«n *
Institut fu«r Biochemie, Bayerische Julius-Maximilians-Universita«t, Biozentrum, Am Hubland, D-97074 Wu«rzburg, Germany
Received 25 September 1998; revised 23 December 1998; accepted 23 December 1998
Abstract
Ribonuclease P is the endonuclease required for generating the mature tRNA 5P-end. The ribonucleoprotein character of this
enzyme has now been proven in most organisms and organelles. Exceptions, however, are still the chloroplasts, plant nuclei and
animal mitochondria where no associated RNAs have been detected to date. In contrast to the known RNA subunits, which
are fairly well-conserved in size and structure among diverse phylogenetic groups, the protein contribution to the holoenzyme is
highly variable in size and number of the individual components. The structure of the bacterial protein component has recently
been solved. In contrast, the spatial arrangement of the multiple subunits in eukaryotic enzymes is still enigmatic. Substrate
requirements of the enzymes or their catalytic RNA subunits are equally diverse, ranging from simple single domain mimics to
an almost intact three-dimensional structure of the pre-tRNA substrate. As an example for an intermediate in the enzyme
evolution, ribonuclease P from the Cyanophora paradoxa cyanelle will be discussed in more detail. This enzyme is unique, as it
combines cyanobacterial and eukaryotic features in its function, subunit composition and holoenzyme topology. ß 1999
Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Ribonuclease P; Ribozyme; Pre-tRNA processing ; Evolution ; Eukaryote; Organelle
Contents
1.
2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacterial RNase P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Variability and conservation in the RNA component . . . . . . .
2.2. Three-dimensional models and the localization of the catalytic
2.3. Substrate binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4. The bacterial RNase P protein . . . . . . . . . . . . . . . . . . . . . . .
RNase P from archaea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Eukaryotic nuclear RNase P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Structure and function of the RNA component . . . . . . . . . . .
4.2. The protein composition of nuclear RNase P . . . . . . . . . . . . .
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* Tel.: +49 (931) 888 4038; Fax: +49 (931) 888 4028; E-mail: [email protected]
0168-6445 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 4 5 ( 9 9 ) 0 0 0 1 4 - 5
FEMSRE 654 12-5-99
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A. Scho«n / FEMS Microbiology Reviews 23 (1999) 391^406
4.3. Substrate speci¢city of nuclear RNase P . . . . . . . . . . . . . . .
Mitochondrial RNase P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. The variability of the RNA subunit . . . . . . . . . . . . . . . . . . .
5.2. The protein subunit of mitochondrial RNase P . . . . . . . . . .
5.3. Substrate recognition by mitochondrial RNase P . . . . . . . . .
6. RNase P from photosynthetic organelles . . . . . . . . . . . . . . . . . . . .
6.1. The enigma of the RNA component in chloroplast RNase P
6.2. RNase P from the Cyanophora paradoxa cyanelle . . . . . . . . .
6.3. Is there a RNA in chloroplast RNase P from higher plants?
7. Summary and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.
1. Introduction
The position-speci¢c endonuclease activity of the
ubiquitous ribonuclease (RNase) P is involved in
processing of tRNA gene transcripts and thus essential for maintaining the functionality of the translation apparatus. This magnesium-dependent enzyme
is composed of RNA and protein in bacteria, archaea, nuclei of yeasts and animals, yeast and plant
mitochondria and in primitive plastids. The bacterial
RNAs are generally active as ribozymes in vitro.
Unlike most other RNA enzymes, RNase P does
not bind its substrate predominantly by base-pairing
interactions. The large number of di¡erent substrates
in a cell require a more complex interplay of structural modules for the exact cleavage site determination, which makes the study of substrate recognition
a revealing enterprise.
Eukaryotic cells contain two or three compartments which are distinct genomic entities: nucleus,
mitochondria and the photosynthetic organelles of
plants. Whereas the nucleus is genetically independent, the latter two have lost genetic information to
this compartment during the evolution. Thus, they
are depending on the import of proteins and in
some cases also of RNA encoded in the nucleus.
The localization of their subunit genes, which may
be encoded on either the organelle or nuclear genome, implies a coordinated expression and possibly
import of certain subunits. An increased knowledge
of organellar RNase Ps is thus not only of interest
for biochemical reasons but will allow a better
understanding of the complex mutual interplay and
regulatory events that are involved in the organelle
biogenesis in eukaryotic cells and may also give in-
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398
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402
sight into the evolution of an ancient RNA enzyme
at the molecular level.
A number of reviews dealing primarily with the
structure and function of bacterial RNase P have
appeared recently [1^5]. After a brief survey of our
current understanding of bacterial RNase P, this paper will then concentrate on novel results, with a
focus on di¡erent aspects of the eukaryotic enzymes,
including the most recent data on the function and
subunit composition of the yeast and human nuclear
enzymes and on the RNA structure of a plastid
RNase P. Naturally, such a snapshot of current
knowledge can never be complete. I apologize in
advance for every recent publication which could
not be discussed here.
2. Bacterial RNase P
2.1. Variability and conservation in the RNA
component
The RNA component of RNase P from bacteria is
certainly the most thoroughly studied. Sequences
from distantly related bacterial phyla, which are encoded by the rnpB gene and vary in length roughly
between 350 and 450 nucleotides, show only little
similarity. Only two longer stretches of sequence
conservation can be recognized, in addition to several short patches [6]. Covariation analysis of sequences covering the whole range of phylogenetic
groups has made the de¢nition possible of a universally conserved minimal consensus structure for these
catalytically active RNAs (Fig. 1, [7]): the two conserved sequence stretches constitute one of the two
FEMSRE 654 12-5-99
A. Scho«n / FEMS Microbiology Reviews 23 (1999) 391^406
393
Fig. 1. Comparison of A- and B-type bacterial RNase P RNAs. Only a schematic representation of the two-dimensional consensus structures is given. P18 and P19, which are not universally conserved, are drawn in gray. The minimum number of base pairs found in each
helical element is represented by heavy lines and variations in the helix length are indicated in gray. The long range interactions P4 and
P6 are connected by thin lines; the sequence of the CCA binding motif in L15 is given in bold letters. Paired elements are numbered
starting from the 5P-end of the RNA [7]. For a better clarity of the ¢gures, only those elements di¡erent from the A-type are numbered
in the B-type structure.
long range base-pairings (P4), the other (P6) connects the hinge region between P5 and P7 to the
distal end of the P15-P16 domain. A further analysis
has led to the identi¢cation of additional tertiary
interactions between short, dispersed structural elements within the RNA [8^10].
All known bacterial sequences fall into two wellde¢ned structural classes ([11], Fig. 1B). Class A,
with Escherichia coli M1 RNA as the prototype, is
represented by the various groups of Gram-negative
and high GC Gram-positive bacteria, whereas
class B structures are mostly restricted to the low
GC Gram-positives, with Bacillus subtilis P RNA
as the prototype. The major di¡erences between the
two classes are the exact position of P6 and the extended P10.1 which may stabilize the ribozyme struc-
ture by interacting with the tetra-loop closing P12
[12].
2.2. Three-dimensional models and the localization of
the catalytic core
Based on a combination of solvent accessibility
and crosslinking experiments, three-dimensional
models for the core region of the RNase P RNA
ribozymes from E. coli and B. subtilis were obtained
which corroborate the covariation data [13^17].
Although they deviate from each other in some details, the localization and orientation of the ribozyme
core region is similar in all models. The central element is helix P4, composed of the two distantly located stretches of sequence conservation. Speci¢c
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A. Scho«n / FEMS Microbiology Reviews 23 (1999) 391^406
age site in the ES complex. Helices P7^P11 make up
the third domain, a complex four way junction. The
pre-tRNA `hinge' is bound to this region, providing
the correct orientation of the substrate phosphate
(see below).
2.3. Substrate binding
Fig. 2. A three-dimensional model of the core region of bacterial
RNase P RNAs. Only a summary is given of the current, experimentally supported models. Helical elements of RNase P RNA
core are drawn as cylinders with the appropriate length and diameters and numbered as in [13]. Helices P6, P8, P10, P17 and
P18, which are either located in the background or are not directly involved in the ribozyme function, have been omitted from
the ¢gure to improve the clarity. The positions where the cyanelle RNase P RNA deviates from the consensus sequence is indicated by arrows. The pre-tRNA is shown as backbone representation, with the CCA-end identi¢ed by lettering and the
cleavage site marked with an asterisk.
The diversity of the pre-tRNAs possesses interesting questions concerning substrate recognition. As
no obvious sequence similarities exist between the
5P-£anks and the full complement of tRNAs present
in each organism show a considerable sequence variation, substrate binding must rely on the recognition
of the overall tertiary structure, non-speci¢c binding
of accessible pre-tRNA regions, speci¢c interaction
with the few absolutely conserved nucleotides in each
tRNA or a combination thereof.
A pronounced in£uence of the pre-tRNA 3P-terminus on the product formation had already been detected for A- and B-type RNase P ribozymes in early
studies. A conserved motif responsible for binding
was later identi¢ed by both genetic and biochemical
means. Removal or mutation of the CCA-end leads
phosphates in this helix are important for binding of
(catalytic?) Mg2‡ -ions and the acceptor stem of the
substrate [18,19], thus supporting the idea that the
most conserved part of the RNA indeed may constitute the catalytic core. In a simpli¢ed view of the
spatial arrangement of RNase P RNA (Fig. 2), P4 is
surrounded by three structural domains which are
anchored to each other mostly by tertiary interactions, like tetra-loop-helix docking [10,15^17]. One
domain consists of helices P1^P3 in a quasi-coaxial
arrangement. Its function in the ribozyme is not
known. The second domain is composed of helices
P15^P18 and is functionally well de¢ned: J16-15 (or
L15 in the B-type RNAs) contains the binding site
for the pre-tRNA CCA-end. Recent data suggest
that this substrate domain may also be involved in
the binding of Mg2‡ -ions participating in the catalytic step [20]. Other metal ions possibly important
for catalysis are coordinated by the phosphate at the
cleavage site itself [21^23]. The nucleotides linking
the CCA binding domain to the remainder of the
ribozyme (J5-15 and J18-2) are adjacent to the cleav-
Fig. 3. Binding of the substrate CCA-end by RNase P RNA.
The proposed interactions between the pre-tRNA 3P-end and
RNase P RNAs containing the canonical GGU motif in L15 are
shown. The purine base found at the discriminator position in
most tRNAs is represented by R; the RàU pair is drawn as a
wobble. Only the 7 bp of the acceptor stem (corresponding to
the minimal substrate) are included in the schematic representation of the pre-tRNA. The remainder of the tRNA body, which
is dispensable for RNase P recognition, is sketched as a stemloop in gray. The variability in the length and structure of P16
and the closing loop is also indicated by shading.
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A. Scho«n / FEMS Microbiology Reviews 23 (1999) 391^406
395
Fig. 4. Two-dimensional representation of RNase P RNAs from the cyanobacteria P. marinus (A) and P. hollandica (B), where the putative CCA binding motif is boxed. Note that the base pair closing L19 in (B) is inversed compared to [39], where an erroneous structure
had been printed.
to an increase in KM , indicating a contribution to the
formation of the enzyme-substrate complex [24^29].
However, the reduced turnover of substrates lacking
the 3P-terminus is alleviated by the protein subunit in
the holoenzyme [1]. The proposed ribozyme-substrate interaction consists of two standard WatsonCrick pairs and an additional pair, which could be
either Watson-Crick or wobble (Fig. 3). It involves
the CC and the proximal single-stranded purine base
(the so-called `discriminator') of the pre-tRNA 3Pend and the sequence 5P-GGU-3P in J16/15 (or L15
in the B-type RNAs) in RNase P RNA. NMR studies and molecular modelling support this mode of
CCA binding [30,31].
It is interesting to note that this substrate binding
motif is present in most, but not all, of the bacterial
lineages examined. Its presence correlates to a certain
degree with the presence of the CCA-end in tRNA
genes in the respective organisms. All tRNA genes of
E. coli, and their majority in B. subtilis, encode the
CCA. Both organisms contain the GGU motif in
their RNase P RNA. In contrast, the tRNA genes
from chloroplasts and other photosynthetic organelles, as well as from cyanobacteria (their phylogenetic ancestors), usually do not encode the CCA-terminus [32^35]. In striking accordance, RNase P
RNAs from primitive plastids and most cyanobacteria are lacking the otherwise highly conserved binding motif [32,36^40], (Fig. 4). Quite unexpectedly,
the cleavage reaction performed by the ribozyme
from the cyanobacterium Prochlorococcus marinus
shows a pronounced dependence on the presence of
the CCA-terminus, even though the GGU motif is
missing from this RNase P RNA [40]. The way in
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which the substrate 3P-end is recognized in this case
is not yet understood.
Although the pre-tRNA 5P-£ank contacts the ribozyme in proximity to the catalytic center, its role in
the active complex is not well-de¢ned [16,25]. The
nucleotides surrounding the cleavage site are also
involved in substrate binding, possibly via their exocyclic amino groups [22,28,41].
A participation of the T-stem and -loop domain in
substrate binding to the RNase P ribozyme has been
established for E. coli and B. subtilis. The T-stem
crosslinks to P8 in the cruciform structure. Certain
nucleotides in the T-loop and in the stem itself seem
to dock onto the ribozyme via their 2P-OH groups
[17,42,43]. The overall structure and parts of the sequence of the cruciform P7^P11 are well-conserved
in the bacterial RNase P RNAs. Among the few
exceptions are those from cyanobacteria and plastids
[6,43]. Interestingly, a tRNAGlu of unique structure
and dual function exists only in the cyanobacterial
line of descent. It deviates from the conserved Tstem sequence of other tRNAs by a change from
the terminal G-C to an A-U pair [35,44]. It is tempting to consider the changes in the cruciform region
of the ribozyme as an adaptation to this unusual
variation in the substrate structure, although the
substrate turnover for pre-tRNAGlu by cyanobacterial RNase P RNAs is slightly reduced compared to
a mutant containing the `canonical' T-stem [39,40].
The exact localization of the cleavage site in E. coli
RNase P is thus primarily determined by the 3P-end
and by the combined length of the acceptor- and Tstem-helices which terminate in the hinge of the Lshaped tRNA structure [1,3]. Even bipartite acceptor
stem mimics are cleaved correctly, lending this system an e¤cient tool for speci¢c mRNA inactivation
in vivo [45,46].
2.4. The bacterial RNase P protein
Although bacterial RNase P RNA is a ribozyme
in vitro, the presence of a functional protein subunit
is essential for the enzymatic activity in vivo [3].
Bacterial RNase P proteins are encoded by the
rnpA gene, which together with rpmH (encoding ribosomal protein L34) constitutes a well-conserved
operon. In all cases where this operon has been identi¢ed, it is located close to the dnaA region. Excep-
tions to this conserved gene arrangement have been
found in two cyanobacteria, two purple bacteria, and
a mycoplasma, whereas it is located at a distance of
10 kbp in Mycoplasma pneumoniae, the rpmH-rnpA
operon is about 500 kbp away from dnaA in the
cyanobacterium Synechocystis PCC 6803. In the other cases, the operon has not yet been localized [47].
The protein component of the holoenzyme is only
120^140 amino acids long, with an unusual abundance of aromatic and basic residues which constitute the only short patch of signi¢cant sequence conservation between the proteins from di¡erent
bacterial species [6]. Despite this lack of similarity,
a holoenzyme activity can be reconstituted from heterologous components, indicating that function,
structure and the mode of binding to the RNA component are conserved in the proteins from divergent
bacterial lineages [48,49]. Even a minimal ribozyme
cut at the junction between the cruciform and core
regions can functionally interact with the protein
[50]. The subunit stoichiometry in the holoenzyme
is 1:1 [51,52].
The observation that the ribozyme requires a signi¢cantly higher ionic strength than the RNase P
holoenzyme led to the assumption that the function
of the protein subunit is merely that of an electrostatic shield. However, the holoenzyme does not
show the strong preference for CCA-containing substrates observed with the ribozyme and it also
cleaves substrates not recognized by the RNA alone,
such as the precursor to 4.5 S RNA, a polycistronic
mRNA, a phage-encoded antisense RNA and the
precursor to tmRNA in E. coli [53^56]. The protein
subunit not only alleviates the strong product inhibition observed in the ribozyme reaction but also extends the substrate range by facilitating binding and
catalysis [46]. Spectroscopic and functional analysis
of protein mutants revealed that certain aromatic
and basic residues are involved in the holoenzyme
formation or substrate binding [57,58].
In the E. coli holoenzyme, the RNA regions protected from the solvent in the presence of the protein
are located in four distinct patches: P3 and J3^4,
part of the cruciform region (J5^7^P8), the base of
P12, J12^13 and P13 and P16 [15,59,60]. Although
no contacts between the substrate and the protein
subunit in the holoenzyme could be demonstrated
directly, the presence of the protein subunit dramat-
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ically increases the a¤nity of the enzyme for pretRNA but not for tRNA, possibly by interacting
with the 5P-£ank [61].
The three-dimensional structure of the protein, as
determined by X-ray crystallography, revealed an
arrangement of three K-helices surrounding a central
L-sheet and three putative RNA binding regions [62].
The ¢rst consists of helix B and the loop connecting
the L-sheet to this helix and exposes the conserved
stretch of basic residues found in all RNase P proteins to the solvent. The second resides in a cleft
between helix A and the L-sheet. It contains several
aromatic residues and is surrounded by basic amino
acids, suggesting that nucleotide binding may occur
via aromatic stacking interactions. The third is a
loop located opposite of the basic loop-helix region.
The few acidic amino acids of the RNase P protein
are clustered here, providing a metal ion binding
pocket and thus suggesting a role in the metal-mediated RNA binding. This overall topology of the
RNase P protein is strikingly similar to that of two
universally conserved proteins of the translational
apparatus, suggesting that it arose early in the protein evolution and thus mediated the conversion
from an all-RNA to an RNP world [62]. The conserved genetic linkage between the RNase P protein
and a ribosomal protein possibly points to the same
evolutionary origin.
3. RNase P from archaea
Like their bacterial counterparts, RNase Ps from
archaea are ribonucleoproteins. Unlike those, however, the biochemical and physical properties of
archaeal holoenzymes indicate that the contribution
of the protein subunit(s) is highly variable in di¡erent species [63^65]. Neither the protein composition
nor the substrate recognition properties of the archaeal enzymes have been investigated to date.
Covariation analysis of archaeal RNase P RNAs
from phylogenetically divergent lineages have allowed the construction of a secondary structure
model similar to the bacterial consensus [66]. The
lack of catalytic activity obvious from these studies
was initially attributed to the minor divergences
from the bacterial structure. However, recent investigations revealed that under more drastic reaction
397
conditions, the RNAs from some but not all phylogenetic lineages can function as ribozymes (Pannucci,
Haas, Hall and Brown, personal communication).
4. Eukaryotic nuclear RNase P
4.1. Structure and function of the RNA component
An intact RNA component is essential for the human and yeast RNase P activity [67^69] and for the
cell viability in yeasts [70,71]. Saccharomyces cerevisiae RNase P RNA is transcribed by polymerase III
from an unusual, externally positioned promoter.
This organisation permits the evolution of the
RNA structure independent from sequence constraints posed by the transcription apparatus [72].
No RNA component could yet be identi¢ed in
nuclear RNase Ps from plants, the slime mold Dictyostelium discoideum, the ciliate Tetrahymena thermophila or from the mold Aspergillus. However, nuclease sensitivity and buoyant density of these
holoenzymes support the hypothesis that they contain an RNA subunit, which may be more or less
accessible to external factors in di¡erent organisms
[73^77].
Considerable progress has been made in developing structural models for RNase P RNAs from
yeasts and vertebrates by phylogenetic covariation
analysis and direct structure determination [78^80].
Although the eukaryotic sequences are highly variable between taxa, a consensus model could be developed which resembles those described for bacterial
and archaeal RNase P RNAs [81]. However, RNase
P RNA genes of di¡erent yeasts can only complement deletion mutants in closely related species [82].
The functional importance suggested by nucleotide
conservation was corroborated by mutational analysis of yeast RNase P RNA in vivo [83]. An RNA
domain essential for the holoenzyme function could
be identi¢ed in these experiments. It corresponds to
J11^12 in the bacterial model and is thus part of the
`universal internal loop' of all RNase P RNAs [81].
Although the exact function of this domain in the
yeast holoenzyme is not clear at present, its structure
in solution is strongly dependent on Mg2‡ -ions, suggesting a role in the metal coordination or substrate
binding near the active center [84,85].
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4.2. The protein composition of nuclear RNase P
The direct identi¢cation of proteins associated
with and essential for eukaryote RNase P activity
has long been hampered by the instability of these
enzymes during puri¢cation. The analysis has been
further complicated by the presence of a second ribonucleoprotein enzyme, RNase MRP. The RNA
component of this exclusively eukaryotic enzyme required for rRNA processing has a cage-shaped core
structure similar to that of RNase P RNA [86].
Although certain proteins co-puri¢ed with RNase
P activity from Schizosaccharomyces pombe [87] and
Aspergillus [77], their functional relation to RNase P
is not clear. Human RNase P reacts with antibodies
from patients su¡ering from the autoimmune disease
scleroderma. Puri¢cation of the HeLa cell enzyme
revealed six proteins correlating with activity, Two
of them (RPP30P and RPP38P) are scleroderma
antigens [88]. In yeast, a genetic analysis has lead
to the discovery of the protein genes POP1^POP4
which are involved in the tRNA and rRNA maturation. Antibodies directed against the corresponding
proteins precipitated yeast and human RNase P activities. Likewise, the scleroderma antibodies react
with RNase P from both sources [89^92]. Disruption
of the yeast gene RPP1 which is homologous to
human RPP30 led to severe defects in tRNA
and rRNA processing, suggesting that these proteins are indeed subunits of the eukaryotic enzyme
[93].
Puri¢cation of the yeast enzyme to apparent homogeneity revealed a total of nine proteins associated with the activity [94]. Four of these proteins
are identical to POP1, 3, 4 and RPP1, previously
identi¢ed by genetic methods. None of them exhibits
a signi¢cant sequence similarity to the known RNase
P proteins from bacteria or yeast mitochondria or
contains any of the known RNA binding motifs.
However, all except POP8P are rich in basic amino
acids. All nine proteins are essential for the cell viability, leading to severe de¢ciencies in the biogenesis
of tRNAs and 5.8 S rRNA. This suspicious link to
RNase MRP is further corroborated by the ¢nding
that eight of the nine protein subunits are shared by
the two enzymes and that the antibodies reacting
with RNase P also precipitate RNase MRP. The
two enzymes are closely associated in macromolecu-
lar complexes and seem to be predominantly localized in the nucleolus of human cells [95]. Although
the lack of ribozyme activity and the direct contacts
between pre-tRNA and RNase P proteins in the eukaryotic enzymes suggest the participation of the
protein complement in the substrate binding or catalysis [96], the exact function of each component of
this multisubunit enzyme still remains to be determined.
4.3. Substrate speci¢city of nuclear RNase P
Most of the currently available data on the substrate speci¢city have been obtained with rather
crude cellular or organelle extracts containing the
full complement of tRNA processing enzymes. The
few exceptions where the puri¢cation of RNase P
allowed a more detailed analysis are the enzymes
from human HeLa cells, Xenopus oocytes, Drosophila cells and wheat germ.
E¤cient cleavage by HeLa RNase P requires not
only the acceptor/T-stem axis including a native
T-loop, but also at least two of the remaining three
tRNA domains (i.e. D-, anticodon- and variable
arm). A minimal substrate where the acceptor- and
T-stem are connected by a short linker is still recognized, but with a much lower e¤ciency [97]. Similar
requirements have been determined for the enzymes
from Xenopus [98] and Drosophila. Cleavage of fullsize substrates by the £y RNase P is not only dependent on the D-loop sequence but also on the
correct interaction between the D- and T-Loop
[99,100]. Like E. coli RNase P, the HeLa cell enzyme
can cleave bipartite substrates in vitro and in vivo.
The minimum requirement in this case is the presence of the complete 3P-half of the tRNA, consisting
of the acceptor-, T-, variable- and anticodon-arm
[101,102].
In processing competent wheat germ extracts,
cleavage at the tRNA termini always precedes intron
removal and splicing. On the other hand, the presence of the intron seems to decrease the rate of end
maturation (reviewed in [103]). Although the processing events at the 5P- and 3P-£anks are fast and
not mutually exclusive, it is clear that 3P maturation
precedes the cleavage by RNase P [74]. The observation that processing of the 5P-end takes place before
cleavage at the 3P-terminus in a carrot cell extract
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may be due to the enrichment of the system for
5P-processing activity [73]. As for the animal systems,
a correct tertiary structure of the pre-tRNA is required for processing by puri¢ed wheat germ RNase
P. Disruption of either the acceptor-, anticodon- or
T-stem in naturally occurring tRNA pseudogenes results in a severe reduction of end-maturation, whereas a change in the more variable extra arm is of
minor in£uence. Studies performed with truncated
or bipartite substrates corroborate the observation
that cleavage is critically dependent on an intact
tRNA structure [74].
5. Mitochondrial RNase P
In addition to the nucleus where most of the genetic information is stored and expressed, eukaryotic
cells contain mitochondria and chloroplasts as energy-generating organelles. According to the endosymbiont hypothesis, these compartments are descendants of free-living bacteria [104]. Accordingly,
they contain their own genomes. During the course
of evolution, genetic information has been transferred to the nucleus to various extents, leading to
a severe reduction in the organelle genome size and
necessitating the import of proteins and in some
cases of RNAs expressed from the nuclear genome.
Although the phylogenetic relationship between organelles and their bacterial ancestors may lead to the
expectation that organelle RNase P might be of the
bacterial type, the possible distribution between two
genetic compartments of the genes encoding such a
multi-subunit enzyme complicates the search for and
analysis of these components.
5.1. The variability of the RNA subunit
Most of the information about mitochondrial
RNase P has been obtained by a genetic and biochemical analysis of S. cerevisiae. Early studies
mapped the tRNA-processing de¢ciency of certain
petite strains to an RNA-encoding gene, RPM1.
The corresponding gene product is the essential
RNA component of RNase P. Although very A-U
rich, the RNA sequence contains the two sequence
stretches conserved in all RNase P RNAs and can be
folded into the typical secondary structure. Interest-
399
ingly, the fragmentation of this RNA during the enzyme puri¢cation does not interfere with the processing activity, suggesting that substrate binding and
catalysis may reside primarily in the protein subunit(s) [105]. Mitochondrial RNase P RNAs from
di¡erent yeasts are quite variable in length, varying
between 140 and 490 nucleotides [106,107]. The corresponding RNA from the mold Aspergillus is 232
nucleotides long [108]. The RNase P RNA encoded
on the mitochondrial genome from the primitive protist R. americana is even more similar to the conserved bacterial structure than those from the fungal
lineage [109]. The characterization of plant mitochondrial RNase P led to the identi¢cation of an
associated RNA fragment. The function of this
RNA and the localization of the corresponding
gene have not been determined [110]. Even RNA
import cannot be excluded in the case of plants. In
contrast to unicellular organisms, the mitochondria
from metazoa in general do not encode an RNase P
RNA [111]. According to biochemical data, human
mitochondrial RNase P does not seem to contain a
RNA subunit [112].
5.2. The protein subunit of mitochondrial RNase P
Yeast mitochondrial RNase P contains a protein
subunit of about 100 kDa. It is the most abundant
protein in enzyme preparations and is required for
the pre-tRNA 5P-end maturation in vivo [113]. The
RPM2 sequence shows no similarity to the protein
components of bacterial or yeast nuclear RNase P
[114]. In addition to pre-tRNA maturation, RPM2P
is involved in the processing of the RNase P RNA
and is required for normal growth even in the absence of mitochondrial translation [115,116]. Di¡erent from S. cerevisiae, several proteins co-purify with
mitochondrial RNase P activity from Aspergillus
[117], however, their functional relation to the
RNase P activity is not clear at present.
5.3. Substrate recognition by mitochondrial RNase P
Most of the studies on substrate requirement have
been performed with crude extracts from human or
plant mitochondria. The two systems are signi¢cantly di¡erent in their gene expression strategies.
Whereas vertebrate mitochondria have extremely
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Fig. 5. Organisation of the genomic region surrounding rnpB. The circular plastomes of P. purpurea (Por), C. paradoxa (Cya) and the liverwort Marchantia polymorpha (Mpo) are represented in linear form, opened within the small single copy region [34,128]. Only a small
portion of the genome is shown for the cyanobacterium P. marinus [40]. (Inverse) repeats are drawn as open boxes, rRNA operons as
black bars. The transcription is from left to right for the genes above the line representing the chromosomes, which have been truncated
arbitrarily (dotted lines). The genes missing in Odontella are shaded in gray in the Porphyra plastome, which is otherwise quite similar in
organisation. Some of the orfs close to Porphyra rnpB are only indicated by short bars without gene names. Abbreviations are: H (trnH),
R (trnR), A (psbA), B^D (petB^petD operon). The latter two have been included for an easier orientation on the plastid sequences, their
location on the P. marinus genome is not known.
small, compact genomes transcribed as one large
polycistronic RNA for each strand, those from
plants have rather large genomes containing long
intergenic regions. The primary transcripts of animal
mitochondria have to be processed to obtain functional RNA species. tRNAs, which are encoded directly adjacent to mRNAs, serve as signals for the
processing system. Another peculiarity is the occurrence of tRNAs with a highly divergent structure.
Frequently, the T-loop sequence deviates from the
consensus or some structural domains like the Dstem are missing completely [35]. Human mitochondrial RNase P is obviously adapted to such a complex variety of substrates and prefers homologous
substrates over bacterial or nuclear pre-tRNAs. 5Pcleavage tends to occur before 3P-maturation [118].
An unusual way of processing has been determined
for a tRNASer which not only lacks the D-domain
but is also directly adjacent to two other pre-tRNAs.
5P-maturation of pre-tRNASer is not performed by
RNase P in this case but occurs by 3P-processing of
the upstream tRNA [119].
In contrast to the vertebrate system, plant mitochondrial transcripts frequently require editing to
gain their function [120]. Primary transcripts which
cannot fold into the three-dimensional tRNA structure are not processed and usually have to be edited
to correct the corresponding base mismatches [121^
123]. In some cases, however, such mismatches are
not removed by editing and the structural deviations
of the pre-tRNA are tolerated by the processing enzymes [124]. Generally, 5P-processing precedes cleavage at the 3P-end in plant mitochondria. Since recognition by RNase P is dependent on an intact
substrate structure, editing is required as the ¢rst
step of tRNA maturation [125].
6. RNase P from photosynthetic organelles
Photosynthetic organelles are phylogenetically-derived from cyanobacteria [104]. During evolution,
the plastome size has been reduced to about onetenth compared to their free-living ancestors, with
a pronounced tendency in the phylogenetically
younger species to relocate certain genes to the nucleus. The similarity between cyanobacteria and plastids, both in metabolism and gene structure, provokes the assumption that RNase P from both
sources may be similar in composition. As detailed
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below, this is true for the `primitive' plastids from
some non-green algae. However, the enzyme from
higher plants may be quite di¡erent.
6.1. The enigma of the RNA component in chloroplast
RNase P
Several lines of evidence, such as protein-like density and insensitivity against micrococcal nuclease,
have led to the conclusion that spinach chloroplast
RNase P does not contain an essential RNA component [126]. The observation that this enzyme is not
signi¢cantly inhibited by the presence of a phosphothioate at the substrate bond even suggests a di¡erent cleavage mechanism than in the bacterial type
ribozyme [23,127]. As the plastomes of green algae
or higher plants do not contain any signi¢cant sequence similarity to bacterial RNase P RNAs (summarized in [103]), any RNA component would have
to be imported, a process which has not yet been
demonstrated for photosynthetic organelles.
6.2. RNase P from the Cyanophora paradoxa
cyanelle
The photosynthetic organelle (cyanelle) of C. paradoxa occupies a bridge position in the plastid evolution, combining properties of free-living cyanobacteria and modern chloroplasts. The cyanelle genome
encodes a RNase P RNA which conforms to the
A-type bacterial structure and is an integral and essential component of the organellar RNase P
([36,128], see Fig. 1A). The observed fragmentation
of cyanelle RNase P RNA upon enzyme puri¢cation
does not signi¢cantly a¡ect the holoenzyme activity,
a situation which is reminiscent of the yeast mitochondrial enzyme [105]. In contrast, the sensitivity of
the RNase P activity against treatment with micrococcus nuclease indicates that a `minimal core' of
RNA is obviously required for the cyanelle RNase
P function [36].
Cyanelle RNase P RNA deviates from the consensus at two positions located in close proximity to the
catalytic center in three-dimensional models of the
bacterial RNAs Fig. 2. The lack of catalytic activity
renders it similar to eukaryotic and some archaeal
RNase P RNAs which closely resemble the consensus but are no ribozymes ([78], Pannucci, Haas, Hall
401
and Brown, personal communication). The protein
contribution in the cyanelle holoenzyme is much
higher than in the bacterial enzymes (Cordier and
Scho«n, unpublished). The idea that the protein component took over some functions of the RNA, as
postulated for the eukaryotic enzymes [96], is thus
particularly striking. No sequence corresponding to
the bacterial rnpA genes is present even on the most
primitive plastid genomes [32^34], indicating that either the protein subunit of chloroplast RNase P is
di¡erent from the bacterial sequence or that its gene
has been relocated to the nuclear compartment during the plastid evolution.
6.3. Is there a RNA in chloroplast RNase P from
higher plants?
The discovery of a RNase P RNA in an organelle
that is considered to be a remnant of evolution,
again raises the question whether RNase P from
`modern' chloroplasts contains a RNA component.
The only other plastid RNase P RNA gene known to
date is that from the red alga Porphyra purpurea [32].
Strikingly, the general arrangement in the rnpB regions is quite similar in Cyanophora, Porphyra and
the cyanobacterium P. marinus Fig. 5. In the cyanelle, two tRNA genes are directly £anking rnpB
on the opposite DNA strand [128]. In the red alga,
several protein genes are interspersed between the
RNA genes. In the cyanobacterium, only the head
to head arrangement of rnpB and one tRNA is
present. In the phylogenetically younger diatom
alga Odontella, the gene orientation is similar to Porphyra, but rnpB and two £anking protein genes have
been deleted [34]. The three plastids discussed here
are members of a di¡erent phylogenetic branch than
the chloroplasts of green algae and higher plants,
where the arrangement of the corresponding genes
is quite variable. Thus, several major recombination
events which occurred after the divergence of the two
lineages must have led to the disruption of the rnpB
region in modern chloroplasts. No complete plastome sequences are yet available for the most primitive members of this branch. Thus, to learn more
about the fate of rnpB and about the RNase P subunit composition in chloroplasts, a systematic analysis of this enzyme from primitive photosynthetic
organisms will be necessary.
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7. Summary and outlook
Signi¢cant progress has been made in the understanding of structure-function relationships in the
bacterial RNase P ribozyme and its protein component. In the eukaryotic systems, the protein complement is highly variable between cellular compartments, the number of subunits varying between one
in yeast mitochondria and nine in the nucleus. The
detailed characterization of all subunits and comparative analysis of di¡erent organisms and organelles
will certainly reveal novel insights into complex
structure-function relationships and also into the
evolution of this ribonucleoprotein enzyme.
[10]
[11]
[12]
[13]
[14]
Acknowledgements
[15]
The research on RNase P in my laboratory is
supported by grants from the Deutsche Forschungsgemeinschaft.
[16]
[17]
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