A structural and functional study of plastid RNAs homologous to catalytic bacterial RNase P RNA

Gene 321 (2003) 47 – 56 www.elsevier.com/locate/gene

A structural and functional study of plastid RNAs homologous to catalytic bacterial RNase P RNA Jesu´s de la Cruz1, Agustı´n Vioque * Instituto de Bioquı´mica Vegetal y Fotosı´ntesis, Universidad de Sevilla-CSIC, Centro de Investigaciones Cientı´ficas Isla de la Cartuja, Avda. Ame´rico Vespucio s/n, Sevilla E-41092, Spain Received 7 March 2003; received in revised form 8 July 2003; accepted 22 July 2003 Received by A. Roger

Abstract Ribonuclease P (RNase P), the ubiquitous enzyme required for 5Vmaturation of transfer RNA, is a ribonucleoprotein containing an essential RNA subunit. This RNA (P RNA) is the catalytic component of RNase P in Bacteria and some Archaea. A putative P RNA is encoded in the chloroplast genome of three algae: Cyanophora paradoxa, Porphyra purpurea and Nephroselmis olivacea. In no case, the P RNAs from the plastids were active in vitro in conditions where bacterial and some archaeal P RNAs are functional. By using lead – ioninduced hydrolysis, we conclude that the catalytic deficiency is most likely due to the perturbation of the global structure of the plastid P RNAs compared to the bacterial counterpart. As a consequence, the plastid P RNAs are unable to bind to the precursor tRNA substrates. We discuss these results in the context of plastid and RNase P evolution. D 2003 Elsevier B.V. All rights reserved. Keywords: Chloroplast; Cyanophora paradoxa; Porphyra purpurea; Nephroselmis olivacea; Synechocystis

1. Introduction In all organisms, tRNAs are synthesized as precursors (pre-tRNAs) that are processed at both 5Vand 3Vends. Ribonuclease P (RNase P) is an ubiquitous enzyme that is essential for generating the mature 5Vend of tRNAs by a single endonucleolytic cleavage on pre-tRNAs (Altman and Kirsebom, 1999). In Bacteria, RNase P is a ribonucleoprotein complex composed of two subunits: a large (350 – 450 nucleotides) RNA (P RNA) encoded by the rnpB gene and a small (ca. Abbreviations: bp, base pair; EDTA, ethylenediaminetetraacetic acid; gifA, glutamine synthetase inactivating factor A gene; HEPES, 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid; lrtA, light repressed transcript gene; P protein, RNase P protein subunit; P RNA, RNase P RNA subunit; PCR, polymerase chain reaction; pre-tRNA, precursor transfer RNA; RNase P, ribonuclease P; rnpA, RNase P protein gene; rnpB, RNase P RNA gene; tRNA, transfer RNA. * Corresponding author. Tel.: +34-954-489-519; fax: +34-954-460-065. E-mail address: [email protected] (A. Vioque). 1 Present address: Departamento de Gene´tica, Facultad de Biologı´a, Universidad de Sevilla, Reina Mercedes 6, E-41012 Sevilla, Spain. 0378-1119/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-1119(03)00831-X

14 kDa) basic protein (P protein) encoded by the rnpA gene. Both subunits are essential for cell viability and required for optimal in vitro activity in reactions at low magnesium and low ionic strength. However, P RNA is catalytically active in vitro in the absence of P protein under high magnesium and high ionic strength conditions. Therefore, bacterial P RNA is a bona fide ribozyme (for a review, see Altman and Kirsebom, 1999). The RNase P of Archaea and the nucleus and mitochondria of Eukarya are also ribonucleoproteins. The RNA subunits from the different phyla show only small patches of sequence conservation, but retain similar structural features to bacterial P RNA (Chen and Pace, 1997). In contrast to the bacterial type, eukaryotic nuclear RNase P contains at least 9– 10 proteins (Xiao et al., 2001). Recent data indicate that archaeal RNase P contains an eukaryal-like P protein set (Hall and Brown, 2002). It has been demonstrated that P RNA from some archaeal species is also catalytically active in the absence of P protein, under extreme ionic conditions, but to a lesser extend than its bacterial counterpart (Panucci et al., 1999). However, experimental conditions that would allow catalysis by the nuclear P RNAs have not been defined

48

J. de la Cruz, A. Vioque / Gene 321 (2003) 47–56

so far. The RNA might be defective in substrate binding, being dependent on one or more proteins for enzymatic activity (Brusca et al., 2001). The RNase P from photosynthetic organelles is particularly striking. In chloroplasts of higher plants, there is biochemical evidence that the enzyme may be composed solely of protein (Wang et al., 1988) although there are not enough data to ensure that it lacks RNA. No gene encoding an RNA homologous to the bacterial P RNA has been identified in the several fully sequenced chloroplast genomes from higher plants and most algae, although some authors claim that P RNA-like sequences could be found in the chloroplast genome of maize and other land plants (Collins et al., 2000) (discussed in Section 4.2). Interestingly, at least some chloroplasts retain a recognizable rnpB gene. Examples have been found in all three primary lineages derived from the original endosymbiont (Fig. 1): the cyanelle of the Glaucophyte Cyanophora paradoxa (Shevelev et al., 1995), the chloroplast of the red algae Porphyra purpurea (Reith and Munholland, 1995) and the chloroplast of the green algae Nephroselmis olivacea (Turmel et al., 1999). The RNase P activity of the cyanelle of C.

paradoxa has been experimentally studied. This activity is sensitive to micrococcal nuclease, and the cyanelle P RNA copurifies with the RNase P activity, strongly suggesting that this RNA is a subunit of the cyanelle RNase P (Baum et al., 1996). Moreover, a fully active RNase P holoenzyme can be reconstituted using in vitro synthesized cyanelle P RNA and P protein from the cyanobacterium Synechocystis sp. PCC 6803 (Pascual and Vioque, 1999a). However, cyanelle P RNA has no in vitro activity at any conditions tested, similarly to nuclear P RNA (Baum et al., 1996; Pascual and Vioque, 1999a) (see also Section 3.2). To date, no information is available on the protein components of the cyanelle RNase P, although biochemical studies suggest that the holoenzyme seems to be more similar in protein content to the nuclear eukaryotic enzyme than to the bacterial enzyme (Cordier and Scho¨n, 1999). We are interested in the molecular characterization of RNase P activity from photosynthetic organisms, especially those from cyanobacteria and plastids of eukaryotic microalgae. Herein, we address the question whether or not chloroplast P RNA from N. olivacea and P. purpurea are functional equivalent to its bacterial counterpart. Like the

Fig. 1. Evolutionary relationships of some representative plastid genomes that have been fully sequenced and the distribution of the rnpB gene. The branching order from the Synechocystis outgroup is taken from Martin et al. (2002). The dotted arrow indicates that the root of the tree lies in the branch leading to Synechocystis. Organisms whose plastid encodes an rnpB gene are circled. The distribution of the rnpB suggests that it has been lost independently more than once in the green and red lineages. Note that C. paradoxa is the only algae whose plastid P RNA can reconstitute a functional holoenzyme with the cyanobacterial P protein (Pascual and Vioque, 1999a). Only the bacterial P RNA can function as a ribozyme by itself.

J. de la Cruz, A. Vioque / Gene 321 (2003) 47–56

cyanelle P RNA from C. paradoxa, these P RNAs are not by themselves catalytically active at any of the conditions tested, even under the extreme ionic conditions that make possible activity of archaeal P RNAs. In contrast to cyanelle P RNA, a functional holoenzyme could not be reconstituted with the in vitro synthesized P RNAs from N. olivacea and P. purpurea and the P protein from the cyanobacterium Synechocystis sp. PCC 6803. We conclude that these P RNAs are not active as ribozymes, at least in part due to the inability of the plastid P RNAs to fold to a correct functional conformation similar to the functional P RNA from Synechocystis or other bacteria. As a consequence, the plastid P RNAs are unable to bind to the pre-tRNA substrates.

49

T7 RNA polymerase to generate P RNA from N. olivacea and P. purpurea, respectively. A plasmid (pSET2) containing the coding sequence of the gifA gene from Synechocystis 6803 under the control of a T7 promoter has been described (Garcı´a-Domı´nguez et al., 1999). A plasmid containing the mRNA leader sequence of the lrtA gene from Synechocystis 6803 under the control of a T7 promoter was a gift from Maribel Muro in our lab. Both plasmids were digested with BamHI and used for run-off in vitro transcription with T7 RNA polymerase to generate RNAs of 470 and 360 nucleotides, respectively. Both RNAs were used as P RNA unrelated controls in inhibition experiments (see below). 2.2. RNase P activity assays

2. Materials and methods 2.1. Templates and RNAs Template plasmids for in vitro transcription of the rnpB gene from Escherichia coli, Synechocystis sp. PCC6803 and the cyanelle of C. paradoxa, as well as for in vitro transcription of pre-tRNATyr of E. coli and pre-tRNAGln of Synechocystis have been described (Vioque, 1997; Pascual and Vioque, 1999b). The rnpB genes from the chloroplasts of N. olivacea and P. purpurea were amplified by polymerase chain reaction (PCR) from total DNA, generously provided by Dr. Monique Turmel (Universite´ Laval, Quebec) and Gertraud Burger (University of Montreal), respectively. For N. olivacea, the forward primer (5V-CCGGAATTCTAATACGACTCACTATAGCTATCTCAAGCAAAGCG-3V) contains an EcoRI site and a T7 promoter sequence and overlaps the 5V end of the coding sequence (italics). The reverse primer (5VCGCGGATCCTGCGCAACTACTCAAGAAATAAGCC-3V) contains a BamHI site and an FspI site overlapping the 3’ end of the coding sequence (italics). For P. purpurea, the forward primer (5V-CCGGAATTCTAATACGACTCACTATAGAAAGTAAACGTAGATAGC-3V) contains an EcoRI site and a T7 promoter sequence and overlaps the 5V end of the coding sequence (italics). The reverse primer (5VCGCGGATCCTTTAAAAAGTACTACATAAGCC-3V) contains a BamHI site and a DraI site overlapping the 3Vend of the coding sequence (italics). To facilitate transcription by T7 RNA polymerase, a G was added at the 5Vend of both rnpB genes. The PCR products were purified on a 2% agarose gel, cloned in pGEM-T (Promega), and both strands sequenced to discard clones containing undesired mutations introduced by the PCR procedure. Then, a positive plasmid clone from each rnpB gene was digested with EcoRI and BamHI, a fragment of the expected size was gel purified from each clone and further ligated into pUC19 digested with EcoRI and BamHI to generate pUC19-NEP and pUC19-POR, respectively. pUC19-NEP was digested with FspI, pUC19-POR was digested with DraI, and then both were used as templates for in vitro run-off transcription with

In all enzymatic assays, pre-tRNATyr from E. coli or pretRNAGln from Synechocystis were used as substrates. The substrates were uniformly labeled by in vitro transcription in the presence of [a-32P]CTP or end labeled with [g-32P]ATP and T4 polynucleotide kinase after dephosphorylation with calf intestinal phosphatase. A variety of conditions were used to test the RNase P activity of the plastid RNAs in the absence of protein. Conditions used ranged from 0.1 to 0.5 M of MgCl2 and from 1 to 7 M of different monovalent cations (K+, NH4+, Na+). Reactions were performed for up to 3 h at 37 jC. Positive control assays with the P RNA from Synechocystis were incubated under identical conditions. RNase P holoenzyme reconstitution assays were done exactly as described (Pascual and Vioque, 1999a). Competition assays were done as previously described (Qin et al., 2001). Briefly, they were done in 20 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) – KOH (pH 7.5), 100 mM MgCl2, 1 M NH4Cl, 5% polyethyleneglycol 6000 and contained 5 nM P RNA from E. coli or Synechocystis, 5 nM substrate pre-tRNA and variable concentrations of competitor RNAs. Aliquots of the reactions were mixed at different times with loading dye containing 7 M urea and 20 mM ethylenediaminetetraacetic acid (EDTA). The amount of processing was estimated by separating the reaction products on polyacrylamide/7 M urea gels and quantification with a Cyclone Phosphor System (Packard). 2.3. Hydrolysis with Pb2+ Reactions with Pb2 + were performed essentially as described (Ciesiolka et al., 1994) except that the P RNAs were labeled at the 5Vend with [g-32]ATP and T4 polynucleotide kinase after dephosphorylation with calf intestinal phosphatase. 10,000 –20,000 cpm of labeled P RNAs were heated for 5 min at 70 jC in 50 mM Tris –HCl (pH 7.5), 0.1 M KCl. Then, MgCl2 was added to a final concentration of 10 or 100 mM and incubation continued for 15 min at 37 jC. Cleavage was initiated by the addition of freshly

50

J. de la Cruz, A. Vioque / Gene 321 (2003) 47–56

prepared Pb(OAc)2 to a final concentration of 0.5 mM and incubation continued for 1 –3 min at 37 jC. To stop the reaction, one volume of loading dye containing 10 M urea and 200 mM EDTA was added. The cleavage products were separated in 8% polyacrylamide/7 M urea gels and detected with a Cyclone Phosphor System (Packard). 2.4. Other methods For Southern blotting, total DNA from N. olivacea was extensively digested with BsrGI and separated on a 0.8% agarose gel. DNA was transferred to and immobilized on a Hybond-N+ nylon membrane (Amersham) following standard procedures. Prehybridization, hybridization and washes were also done according to standard procedures. As a probe, a 450 bp EcoRI – BamHI fragment from pUC19NEP, containing the rnpB gene from N. olivacea was used. The probe was labeled with a DNA labeling kit (Ready to Go, Amersham). Hybridization bands were visualized using a Cyclone Phosphor System (Packard).

3. Results 3.1. Distribution of rnpB homologous genes in plastid genomes To date, more than 20 chloroplast genomes have been fully sequenced. From the sequence analysis, it can be concluded that only the cyanelle of the glaucophyte C. paradoxa, the chloroplast of the green algae N. olivacea and the chloroplast of the red algae P. purpurea encode for a bacterial-like rnpB gene. The rest of the available genomes contain neither a bacterial-like rnpB gene nor a sequence that would fit to the minimal consensus found in archaeal and eukaryal P RNAs (but see Section 4.2). In Fig. 1, we show a scheme of the evolutionary relationships of some representative chloroplast from the different lineages whose genome has been sequenced. The distribution of rnpB among chloroplast genomes indicates that there have been multiple losses of this gene among the different chloroplast lineages. A similar pattern of independent parallel gene losses is general for most other plastid genes in the evolution of chloroplasts (Martin et al., 2002). Therefore, we found interesting to analyze the structure and function of the putative plastid P RNAs from C. paradoxa, N. olivacea and P. purpurea. To do so, we constructed templates by PCR amplification from total DNA isolated from N. olivacea and P. purpurea (see Section 2.1). Sequence analysis of the amplified rnpB gene from the plastid of P. purpurea corresponds exactly to the released genomic sequence (Reith and Munholland, 1995). However, sequence analysis of the amplified rnpB from the plastid of N. olivacea did not fully match with the published genomic sequence (Turmel et al., 1999). Sequence differences were found in the following positions: insertion of a

G in position 317, and substitutions U318A, U328G, C357A and C383A (Fig. 2). Three independent clones from three independent PCR reactions were sequenced and they all led to the same sequence, strongly suggesting that the true genomic sequence corresponded to the one we obtained. G317, A318 and A383 are among the few universally conserved nucleotides in bacterial P RNAs (Brown, 1999). G328 is part of helix P2 and, as it base pairs with a C, any change of this nucleotide would disrupt the helix structure. To further confirm that our sequence was the authentic genomic sequence, we performed Southern analysis of total DNA from N. olivacea digested with BsrGI. The presence of an A at position 383 would generate a BsrGI site. The Southern blot indicated the presence of the BsrGI site in the rnpB gene (data not shown), demonstrating that an A was present in the plastid rnpB gene at position 383 and not a C. The formal possibility exists that, because we used as template total N. olivacea DNA and not purified plastid DNA, the product we are amplifying corresponds to a nuclear encoded gene. We find this possibility highly improbable because (i) there are only five differences between the sequence we have found and the published one (Turmel et al., 1999), and they are all very close together in a highly conserved segment of the P RNA; (ii) we always recovered the same sequence from different PCR experiments, which would require preferential amplification of the nuclear encoded sequence over the plastid sequence; (iii) the bands detected in a Southern blot experiment are the bands expected from the chloroplast genomic sequence, and no additional bands that would explain the hypothetical nuclear encoded rnpB gene were observed. In summary, as we have used total DNA from the same N. olivacea strain that was used for sequencing of the chloroplast genome, we conclude that the sequence differences detected are due to errors in the previously published sequence (Turmel et al., 1999). 3.2. The structure of plastid P RNAs The plastid P RNA sequences from C. paradoxa, N. olivacea and P. purpurea are obviously homologous to the bacterial P RNA of type A, and they can theoretically fold in a similar secondary structure (Fig. 2). The secondary structure of the P RNA from the cyanelle of C. paradoxa has even been confirmed experimentally to a great extent (Cordier and Scho¨n, 1999). The three plastid P RNAs share with most cyanobacterial P RNA the absence of a GGU sequence in the P15 loop that is important for substrate binding in E. coli P RNA (Kirsebom and Sva¨rd, 1994). The three plastid P RNAs also share with most cyanobacterial P RNAs an extended P6 helix, which is four base pairs long in most bacterial P RNA but that extend to six-seven base pairs in cyanobacterial P RNAs (Vioque, 1997). In addition, the predicted structures of the plastid P RNAs contain a number a deviation from the consensus (Fig. 2). The cyanelle P RNA from C. paradoxa has an A at position 22 instead of the conserved G, and an

J. de la Cruz, A. Vioque / Gene 321 (2003) 47–56

extra A –U base pair in P5 that have been discussed elsewhere (Baum et al., 1996). Most prominent defects are found in tertiary interactions L14 – P8 and L9 – P1 deduced by covariation (Brown et al., 1996; Massire et al., 1997). In the plastid P RNA from P. purpurea, the interaction L14 –P8 does not fit the consensus established by covariation. Similarly, in the plastid P RNA from N. olivacea, the interactions L9 – P1 and L18 – P8 do not fit the consensus established by covariation. In addition, L9 and L18 from the plastid P RNA from N. olivacea are not the usual GNRA tetraloops found in bacterial P RNA (Brown et al., 1996). Moreover, P2 is only six nucleotides long in the plastid P RNA from N. olivacea. Extension of P2 to seven base pairs would require a noncanonical A –C pair at the base of the helix (A15 –C330). Finally, the three RNAs share a high AT content, which would increase structural instability. Pb2 +-induced cleavage has been used as a sensitive probe for the tertiary folding of RNase P (Ciesiolka et al., 1994). Cleavage occurs mainly in single stranded regions. Here, we have compared the Pb2 + cleavage pattern of the plastid P RNAs with the cleavage pattern of a catalytically proficient P RNA from the cyanobacterium Synechocystis 6803 (Fig. 2). In Synechocystis, the primary Pb2 + cleavage site detected is in the bulge of three nucleotides that separate helix P10 from P11. This is the main cleavage site by Pb2 +, so called site Ia, in E. coli P RNA (Ciesiolka et al., 1994) and in several other P RNAs, included the structurally different P RNA from Bacillus subtilis (Zito et al., 1993). This site is observed in 10 mM MgCl2 but not in 100 mM MgCl2, in agreement with the fact that there is a Mg2 + binding site at this position (Kazakov and Altman, 1991). Other Pb2 + cleavage sites present in E. coli P RNA are found at approximately similar positions in Synechocystis P RNA but are rather weak (Ib, IIb, IIc). Other sites are no detected (IIa, IIbV). The sites corresponding to the P15 –P17 region (III –V) are also present. We also observe some Pb2 + sensitive sites downstream of site V; however, these sites cannot be precisely mapped from the experiments shown in Fig. 2. Cleavage at all these sites is weakly competed by high Mg2 + concentration, opposite to what happens at site Ia. A significant difference between Synechocystis and E. coli is the increased sensitivity to Pb2 + cleavage of the P12 helix in Synechocystis P RNA. P12 is unusually long in Synechocystis P RNA and its secondary structure has not been experimentally analyzed previously. However, the Pb2 +-induced cleavage sensitivity detected is in good agreement with the proposed secondary structure for Synechocystis P12 helix (Vioque, 1997), that was only based on computer prediction, since this extended helix has no equivalent in other P RNAs that could be used for covariation analysis (Vioque, 1997). The more sensitive sites are in the loop that closes the helix and in the bulges (Fig. 2), as expected. But there is also sensitivity to Pb2 + in the terminal base pairs before the loop suggesting that, in the actual structure in solution, the terminal stem is not stable enough and it is rather a large loop. Another clear difference

51

between the P RNAs from Synechocystis and E. coli is the sensitivity to Pb2 + cleavage that appears at the P13 loop (L13; labeled A in Fig. 2) in Synechocystis P RNA that has not been described in E. coli P RNA. The only significant difference that is clearly observed in the Pb2 +-induced cleavage between Synechocystis and the three plastid P RNAs we have assayed (see also Fig. 2) is that site Ia is not present in C. paradoxa and N. olivacea plastid P RNAs, and is weak and insensitive to Mg2 + competition in P. purpurea P RNA. Taking in account that site Ia is the main Pb2 +-induced cleavage site present in Synechocyistis and other functional P RNAs, this points to significant differences in the global tertiary structure between the catalytic Synechocystis P RNA and its nonfunctional in vitro (see Section 3.3) plastid P RNAs. Also, the overall sensitivity to Pb2 +-induced hydrolysis is significantly higher at 10 mM Mg2 + in the plastid P RNAs. This was estimated by comparison of the radioactivity remaining in the full length RNA after incubation with Pb2 +. In different experiments, after 3 min of incubation with Pb(OAc)2 in 10 mM MgCl2, only 40– 60% of the radioactivity was in the full length RNA in the plastid P RNAs compared with Synechocystis P RNA (see Fig. 3). Finally, there is a higher sensitivity to spontaneous hydrolysis of the plastid P RNAs, in particular that of N. olivacea, which precludes analysis of Pb2 + sensitivity at many positions. 3.3. Functional assays of plastid P RNAs Different assays were performed in order to determine if these P RNAs, although structurally different to the P RNA from Synechocystis, were catalytically active. Assays were done using pre-tRNATyr from E. coli or pre-tRNAGln from Synechocystis as substrates, in the presence or absence of P protein from Synechocystis, and under a wide range of experimental conditions (see Section 2.2). In all assays, the plastid P RNAs failed to process pre-tRNAs, even in those reaction conditions described to support activity of some P RNAs from Archaea (Panucci et al., 1999) (data not shown). The catalytic deficiency of plastid P RNAs, most likely results of the perturbation of the global tertiary structure of these RNAs (see Section 3.2), could be in principle due to either a deficiency in substrate binding or in catalysis itself, or to a combination of both phenomena. In order to test substrate binding by the inactive plastid P RNAs, we used an inhibition assay. The reaction mixtures contained a small concentration of pre-tRNA substrate (5 nM), 5 nM of active P RNA from E. coli or Synechocystis 6803 and variable concentrations of the plastid P RNAs. As specificity controls, we used as competitors two unrelated RNAs of similar size to the P RNAs. The assays were done with three different active RNA enzyme– substrate pairs whose Km has been experimentally determined. While the Km for the reaction of E. coli P RNA for pre-tRNATyr from E. coli is around 0.03 AM (Altman and Kirsebom, 1999), the Km

52

J. de la Cruz, A. Vioque / Gene 321 (2003) 47–56

J. de la Cruz, A. Vioque / Gene 321 (2003) 47–56 Fig. 2. Secondary structure models and hydrolysis by Pb2 + of P RNAs from Synechocystis 6803, the cyanelle of C. paradoxa, the plastid of N. olivacea and the plastid of P. purpurea. Secondary structure models and helix labeling are taken from the RNase P database (Brown, 1999). Nucleotides or RNA regions highlighted in red deviate from the bacterial consensus and are discussed in the text. The P RNAs from the three algae plastids contain a non-encoded G (also highlighted in red) as first nucleotide for efficient transcription (see Section 2.1). Nucleotides in the plastid P RNA from N. olivacea that are different from the published sequence are shown in green. The inset shows an alternative secondary structure for the top part of the N. olivacea P RNA. Arrowheads or segments indicate the Pb2 + cleavage sites. The sites equivalent to those observed in E. coli are identified with roman numerals as described for E. coli (Zito et al., 1993; Ciesiolka et al., 1994). Other sites are identified with capital letter. See Section 3.2 for further details. Pb2 + cleavage products were separated on 8% polyacrylamide/7 M urea gels and visualized with a Cyclone Phosphor System (Packard). The different Mg2 + concentrations and times of incubation with Pb2 + are indicated above the gels. Structural elements of the P RNAs are labeled and delimited by vertical bars on the left of the gels. The size of DNA markers run in parallel is also indicated. Under the denaturing conditions used, they migrate like RNA fragments of the same size. 53

54

J. de la Cruz, A. Vioque / Gene 321 (2003) 47–56

Fig. 3. Relative overall sensitivity to Pb2 + of the different RNAs analyzed. The plot represents the amount of radioactivity remaining in the full length RNA from C. paradoxa cyanelle (white circles), N. olivacea chloroplast (white squares) and P. purpurea chloroplast (white triangles) after different times of incubation with Pb2 + in the presence of 10 mM Mg2 +. Note that the data were normalized with respect to the amount of radioactivity remaining in the full length P RNA from Synechocystis after the same incubation times, which is taken as 100% in all cases.

for the reaction of Synechocystis P RNA for the same substrate is 0.14 AM (Vioque, 1997). Finally, the Km for the reaction of Synechocystis P RNA for pre-tRNAGln from Synechocystis is 3 AM (Pascual and Vioque, 1999b). Because the concentration of both substrate and enzyme are below Km, binding of the pre-tRNA by the inactive P RNAs would act as a competitive inhibitor of the active P RNAs (Qin et al., 2001). However, as shown in Fig. 4, large excess of the plastid P RNAs did not inhibit significantly RNase P activity of the enzyme –substrate pairs with low (Fig. 4A) or intermediate Km (Fig. 4B). In contrast, significant inhibition was detected for the enzyme– substrate pair with high Km (Fig. 4C). However, a better (Fig.

4A, 4B) or similar (Fig. 4C) inhibition was observed when a control unrelated RNA, such as the gifA mRNA was used as competitor. This indicates that the plastid P RNAs do not have a better affinity to the pre-tRNA substrate than the RNase P unrelated gifA mRNA. To confirm that the inhibitory effect by the gifA RNA observed in the assay of Synechocystis P RNA with pre-tRNAGln was of a non specific nature, we assayed a second unrelated RNA, corresponding to the non coding leader sequence of the lrtA mRNA. The leader of lrtA mRNA produced an inhibitory effect similar to that observed with the plastid P RNAs and the gifA mRNA (Fig. 4C and data not shown). The inhibitory effect can be caused by binding to either the substrate or the active Synechocystis P RNA. We conclude that the absence of specific affinity for the substrate is at least in part responsible for the absence of catalytic activity of the plastid P RNAs.

4. Discussion 4.1. Biochemical reasons for the catalytic deficit of plastid P RNAs Our results indicate that the plastid P RNAs are not active ribozymes under any of the conditions tested, including those used to show catalytic activity of archaeal P RNAs. In addition, the plastid P RNAs are defective in binding of the substrates, as indicated by the competition assays shown in Fig. 4. It could be argued that the plastid P RNAs have a narrower than usual substrate specificity and would not recognize a bacterial pre-tRNA as substrate. However, holoenzyme reconstituted with cyanelle P RNA from C. paradoxa and Synechocystis P protein is active on both substrates used in this work (Pascual and Vioque,

Fig. 4. Effect of plastid P RNAs on RNase P activity. RNase P activity of E. coli P RNA (A) or Synechocystis P RNA (B, C) was assayed with pre-tRNATyr from E. coli (A, B) or pre-tRNAGln from Synechocystis (C) in the presence of variable concentrations of either P RNA from C. paradoxa cyanelle (white circles), N. olivacea chloroplast (white squares) and P. purpurea chloroplast (white triangles), gifA mRNA (black circles), or lrtA leader RNA (black squares). Assays were done with uniformly labeled pre-tRNA as described in Section 2.2.

J. de la Cruz, A. Vioque / Gene 321 (2003) 47–56

1999a). Moreover, crude extracts from C. paradoxa cyanelle and partially purified fractions have high RNase P activity with pre-tRNATyr from E. coli and pre-tRNAGln from Synechocystis (A. Pascual and A.V., unpublished). These results indicate that while the cyanelle holoenzyme can recognize the substrates, the in vitro synthesized P RNA cannot. Therefore, it must be assumed that the protein components of the three plastid P RNAs studied in this work have an essential role in substrate binding, either because it is directly involved in such binding or indirectly by promoting the correct folding of the RNA subunit. In this respect, it is important to mention that although bacterial P RNA can bind efficiently the substrate, the protein subunit enhances the interaction by specific binding to the 5Vleader sequence of the substrate (Niranjanakumari et al., 1998). One of the primary reasons for the catalytic deficit of plastid P RNAs could be their inability to fold into a functional tertiary structure, as suggested by the Pb2 +induced cleavage sensitivity. Several factors could contribute to this inability, such as the high AT content in plastid P RNAs and their deviation from consensus at positions critical for tertiary interactions. Site Ia is within a highly conserved region of P RNA involved in substrate binding, and whose crystal structure in B. subtilis has been recently resolved (Krasilnikov et al., 2003). Two nucleotides directly involved in interaction with the substrate have been identified very close to site Ia in E. coli and B. subtilis (LaGrandeur et al., 1994). These nucleotides correspond to G106 and A270 in the Synechocystis P RNA sequence. Therefore, we propose that the perturbed structure in this region detected by our Pb2 +induced cleavage experiments in the plastid P RNAs could very well be the cause of their inability to bind to substrates. Finally, we cannot exclude that the inability of in vitro transcribed P RNAs might be due to the absence of post-transcriptional modifications that might be present in vivo in plastid P RNAs and needed for their proper folding. 4.2. Evolutionary considerations It seems that, in the course of evolution, P RNA has lost the ability to bind to the substrate, relying more heavily on the protein component. While the nuclear RNase P activity still retains an RNA subunit involved in the catalytic mechanism but unable to bind the substrate, the chloroplast enzyme of higher plants might have completely lost the RNA subunit, being replaced by a so far unknown protein enzyme that could be related or unrelated to protein subunits of RNA-based RNase P. The plastid P RNAs analyzed in this work might represent intermediates in the process of reduction in the capabilities of P RNA; while they are overall similar to the bacterial P RNA, they probably lack some crucial structural elements that make them deficient in

55

proper folding and substrate binding, requiring the cooperation of essential protein subunits. Although the phylogeny of chloroplasts is not fully resolved, it seems clear that the rnpB gene has been lost independently more than once in the course of chloroplast evolution. Six independent losses are required to explain the distribution of rnpB in plastid genomes in the phylogeny shown in Fig. 1 (Martin et al., 2002). Alternative phylogenies would require four to six parallel losses (Martin et al., 2002). It has been proposed that there is an RNase P-like sequence in the plastid genome from higher plants. This sequence overlaps an ORF of unknown function (ORF29) (Collins et al., 2000). There are no data to suggest that this RNase P-like sequence is a functional RNA equivalent to bacterial P RNA. The overlapping ORF29 is well conserved between cyanobacteria and plants, but not the surrounding region that is part of the hypothetical rnpB gene. This indicates that may be the ORF that is functionally relevant but not the untranslated region that would be part of rnpB. Moreover, the sequence of the putative P4 helix in the proposed P RNA-like sequences deviates from the universal consensus in this helix and has unrelated sequence in different plastids from higher plants (our unpublished analysis). Taken together, these considerations argue against the hypothesis that the plastid sequence analyzed by Collins and collaborators is a true functional equivalent of P RNA. One interesting question is the nature of RNase P in the plastids that lack the rnpB gene. It is possible that an RNA homologous to rnpB is imported from the nucleus in those cases. That would not be the case in higher plant chloroplasts if it is clearly demonstrated that RNase P is a proteinonly enzyme, as has been proposed (Wang et al., 1988). Then, it will be important to know if the different algal lineages that lack a plastid rnpB have also evolved an independent protein-based RNase P. Another question is if the plastid P RNA sequences studied here represent non-functional pseudogenes. In the case of C. paradoxa, there is biochemical evidence that the P RNA is part of the cyanelle RNase P (Baum et al., 1996; Cordier and Scho¨n, 1999). This evidence is lacking in N. olivacea and P. purpurea. Thus, although we cannot discard the fact that the plastid P RNAs from N. olivacea and P. purpurea are derived from pseudogenes, the sequence and predicted structure of the corresponding P RNAs is much better conserved that what would be expected from a nonfunctional pseudogene in fast evolving chloroplast genomes.

5. Conclusion This work is the first study that addresses the question of the functional deficiency of plastid RNAs homologous to bacterial P RNA, and provides evidence that in the absence of the protein component these RNAs cannot properly fold and bind the substrate. Identification of the protein subunits of plastid RNase P will be a significant challenge for the future.

56

J. de la Cruz, A. Vioque / Gene 321 (2003) 47–56

Acknowledgements We are grateful to Gertraud Burger and Monique Turmel for providing DNA from P. purpurea and N. olivacea, respectively, and to Jose´ Carlos Reyes and Maribel Muro for plasmids pSET2 and lrtA mRNA leader, respectively. We thank Gabriel Gutie´rrez for critical reading of the manuscript. J.d.l.C. acknowledges financial support from the Spanish Government (Contrato de Reincorporacio´n, Ministerio de Educacio´n y Cultura). This work was supported by grants from the Human Frontier Science Organization (RG291/1997), Ministerio de Ciencia y Tecnologı´a (BMC2001-3789) and Junta de Andalucı´a (CVI215).

References Altman, S., Kirsebom, L.A., 1999. Ribonuclease P. In: Gesteland, R., Cech, J., Atkins, J. (Eds.), The RNA World. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 351 – 380. Baum, M., Cordier, A., Scho¨n, A., 1996. RNase P from a photosynthetic organelle contains an RNA homologous to the cyanobacterial counterpart. J. Mol. Biol. 257, 43 – 52. Brown, J.W., 1999. The ribonuclease P database. Nucleic Acids Res. 27, 314. Brown, J.W., Nolan, J.M., Haas, E.S., Rubio, M.A.T., Major, F., Pace, N.R., 1996. Comparative analysis of ribonuclease P RNA using gene sequences from natural microbial populations reveals tertiary structural elements. Proc. Natl. Acad. Sci. U. S. A. 93, 3001 – 3006. Brusca, E.M., True, H.L., Celander, D.W., 2001. Novel RNA-binding properties of Pop3p supports a role for eukaryotic RNase P protein subunits in substrate recognition. J. Biol. Chem. 276, 42543 – 42548. Chen, J.L., Pace, N.R., 1997. Identification of the universally conserved core of ribonuclease P RNA. RNA 3, 557 – 560. Ciesiolka, J., Hardt, W.D., Schlegl, J., Erdmann, V.A., Hartmann, R.K., 1994. Lead – ion-induced cleavage of RNase P RNA. Eur. J. Biochem. 219, 49 – 56. Collins, L.J., Moulton, V., Penny, D., 2000. Use of RNA secondary structure for studying the evolution of RNase P and RNase MRP. J. Mol. Evol. 51, 194 – 204. Cordier, A., Scho¨n, A., 1999. Cyanelle RNase P: RNA structure analysis and holoenzyme properties of an organellar ribonucleoprotein enzyme. J. Mol. Biol. 289, 9 – 20. Garcı´a-Domı´nguez, M., Reyes, J.C., Florencio, F.J., 1999. Glutamine synthetase inactivation by protein – protein interactions. Proc. Natl. Acad. Sci. U. S. A. 96, 7161 – 7166. Hall, T.A., Brown, J.W., 2002. Archaeal RNase P has multiple protein subunits homologous to eukaryotic nuclear RNase P proteins. RNA 8, 296 – 306. Kazakov, S., Altman, S., 1991. Site-specific cleavage by metal ion cofactors and inhibitors of M1 RNA, the catalytic subunit of RNase P from Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 88, 9193 – 9197.

Kirsebom, L.A., Sva¨rd, S.G., 1994. Base pairing between Escherichia coli RNase P RNA and its substrate. EMBO J. 13, 4870 – 4876. Krasilnikov, A.S., Yang, X., Pan, T., Mondragon, A., 2003. Crystal structure of the specificity domain of ribonuclease P. Nature 421, 760 – 764. LaGrandeur, T.E., Huttenhofer, A., Noller, H.F., Pace, N.R., 1994. Phylogenetic comparative chemical footprint analysis of the interaction between ribonuclease P RNA and tRNA. EMBO J. 13, 3945 – 3952. Martin, W., Rujan, T., Richly, E., Hansen, A., Cornelsen, S., Lins, T., Leister, D., Stoebe, B., Hasegawa, M., Penny, D., 2002. Evolutionary analysis of arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus. Proc. Natl. Acad. Sci. U. S. A. 99, 12246 – 12251. Massire, C., Jaeger, L., Westhof, E., 1997. Phylogenetic evidence for a new tertiary interaction in bacterial RNase P RNAs. RNA 3, 553 – 556. Niranjanakumari, S., Stams, T., Crary, S.M., Christianson, D.W., Fierke, C.A., 1998. Protein component of the ribozyme ribonuclease P alters substrate recognition by directly contacting precursor tRNA. Proc. Natl. Acad. Sci. U. S. A. 95, 15212 – 15217. Panucci, J.A., Haas, E.S., Hall, T.A., Harris, J.K., Brown, J.W., 1999. RNase P RNAs from some Archaea are catalytically active. Proc. Natl. Acad. Sci. U. S. A. 96, 7803 – 7808. Pascual, A., Vioque, A., 1999a. Functional reconstitution of RNase P activity from a plastid RNA subunit and a cyanobacterial protein subunit. FEBS Lett. 442, 7 – 10. Pascual, A., Vioque, A., 1999b. Substrate binding and catalysis by ribonuclease P from cyanobacteria and Escherichia coli are differently affected by 3V-terminal CCA in precursors of tRNA. Proc. Natl. Acad. Sci. U. S. A. 96, 6672 – 6677. Qin, H., Sosnick, T.R., Pan, T., 2001. Modular construction of a tertiary RNA structure: the specificity domain of the Bacillus subtilis RNase P RNA. Biochemistry 40, 11202 – 11210. Reith, M., Munholland, J., 1995. Complete nucleotide sequence of the Porphyra purpurea chloroplast genome. Plant Mol. Biol. Rep. 13, 333 – 335. Shevelev, E.L., Bryant, D.A., Lo¨ffelhardt, W., Bohnert, H.J., 1995. Ribonuclease-P RNA gene of the plastid chromosome from Cyanophora paradoxa. DNA Res. 2, 231 – 234. Turmel, M., Otis, C., Lemieux, C., 1999. The complete chloroplast DNA sequence of the green alga Nephroselmis olivacea: insights into the architecture of ancestral chloroplast genomes. Proc. Natl. Acad. Sci. U. S. A. 96, 10248 – 10253. Vioque, A., 1997. The RNase P RNA from cyanobacteria: short tandemly repeated repetitive (STRR) sequences are present within the RNase P RNA gene in heterocyst-forming cyanobacteria. Nucleic Acids Res. 25, 3471 – 3477. Wang, M.J., Davis, N.W., Gegenheimer, P., 1988. Novel mechanisms for maturation of chloroplast transfer RNA precursors. EMBO J. 7, 1567 – 1574. Xiao, S., Houser-Scott, F., Engelke, D.R., 2001. Eukaryotic ribonuclease P: Increased complexity to cope with the nuclear pre-tRNA pathway. J. Cell. Physiol. 187, 11 – 21. Zito, K., Huttenhofer, A., Pace, N.R., 1993. Lead-catalyzed cleavage of ribonuclease P RNA as a probe for integrity of tertiary structure. Nucleic Acids Res. 21, 5916 – 5920.