Chloroplast ribosomal proteins and their genes

Plant Science, 72 (1990) 1--12 Elsevier Scientific Publishers Ireland Ltd.

1

Review Article

Chloroplast ribosomal proteins and their genes R. Mache Laboratoire de Biologie Mol~culaire V~g~tale, Universit~ Joseph Fourier, URA CNRS 1178, BP 53 F-38041 Grenoble Cedex (France) (Received April 6th, 1990; revision received July 10th, 1990; accepted July 13th, 1990)

Recent works on chloroplast ribosomal proteins and their genes are reviewed. Following topics are included: prokaryotic features; organization and expression of chloroplast-encoded ribosomal protein genes in land plants and in Algae; nuclear encoded ribosomal protein genes; assembly of chloroplast ribosomes; hypothesis on gene transfer from chloroplast genome to nucleus.

Key words: chloroplast; ribosomal proteins; genes; nuclear genes

The first evidence for the existence of chloroplast ribosomes was obtained almost 30 years ago by Lyttleton [1]. Since then, much work has been done to characterize the plastid translational apparatus (for reviews see Refs. 2--4). This review will be focused mainly on plastid ribosomal proteins. Translational factors and the biogenesis of ribosomal proteins in relation with plant development will not be considered here. The genetic system of the chloroplast including the translational apparatus is of a procaryotic type and it is interesting to determine how this translational apparatus has diverged from bacteria. Consequently, chloroplast ribosomal proteins (r-proteins) will be compared to Escherichia coli r-proteins. Whenever possible, r-proteins will be named according to their E. coli homologues. In some cases where the homology is not known or is not relevant, r-proteins will be designated as reported by authors. Number, Site of Synthesis

Establishing the number of r-proteins present in the chloroplast ribosome was the subject of investigations several years ago and the results were previously reviewed [2]. Generally, chloroplast ribosomes contain about the same number of r-

proteins as the ribosomes of E. coli. This point was studied in detail using different 2D-electrophoretic systems. In spinach [5], in tobacco [6] and in Euglena [7] it was found that 33--35 r-proteins are present in the 50S and 22--24 r-proteins in the 30S ribosomal subunits. An exception is Chlamydomonas reinhardtii which was found to contain 31 r-proteins in the small subunit [8] whilst the large subunit contains 33 r-proteins. Chloroplast r-proteins are encoded within two genetic compartments, the chloroplast and the nucleus. The complete sequences of the chloroplast genomes from a monocot [9] a dicot [10] and a lower plant [11] reveal the presence of 20 genes coding for putative r-proteins detected by their homology to E. coli r-proteins. Eleven genes in Marchantia polymorpha [11] or 12 genes in rice and tobacco [9,10,12] code for 30S r-proteins and 8 or 9 genes code for 50S r-proteins. This represents about one third of the total number of rproteins in the chloroplast ribosome and fits well with the number of r-proteins synthesized in chloroplasts [13--15]. Most of the r-protein genes encoded in the chloroplast genome are expressed transcriptionally and translationally. Nevertheless exceptions may exist as observed from studies on the expression of the rp123 gene [16].

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Modifications Chloroplast ribosomal proteins may be modified posttranscriptionally but modifications are not necessarily the same as in E. coli. Thus, in spinach, the N-terminal amino acid of the 12 protein is not a formyl-Met [16,17] but a methylated alanine [17]. In E. coli, six r-proteins are methylated but not the L2. Phosphorylation of plastid r-proteins has been observed by two different groups [15,18] and phosphorylation was shown to occur on serine residues [18]. The same r-proteins are found to be phosphorylated when experiments are performed with isolated chloroplasts or with an in vitro system using isolated chloroplast ribosomes and a cAMP independent protein kinase [18]. In eukaryotic cells the phosphorylation of the ribosomal protein $6 is correlated with an increase in the rate of protein synthesis [19]. In E. coli no r-proteins were found to be modified by protein kinase. Therefore, it would be interesting to know whether the phosphorylation of r-proteins in plastids has regulatory function(s).

Protein Diversity Among Plants As ribosomes are very conserved structures the question arises whether r-proteins vary within closely related species of plants and in particular within one genus. Some data have been obtained that relate to this question with Nicotiana species. Chloroplast r-proteins from N. tabacum (western hemisphere), N. gossei and N. excelsior (Australia) have been compared by 2D-gel electrophoresis [20]. Limited differences in migration were detected for some r-proteins suggesting only little variation. This conclusion is confirmed by the analysis at the sequence level of the rpl2 gene in N. debneyi [21] (sequence corrected by one insertion) and in N. tabacum [22]. In Chlamydomonas Randolph-Anderson et al. [23] found that the interfertile species C. reinhardtii and C. smithii possessed a pair of proteins in each subunit that differ in charge only. By comparison of the number of nucleotide substitutions in the rpl2 gene with those of the rbcL and atpB genes in a different plant taxa, Zurawski and Clegg [24] found

large differences between the two groups of genes. This could mean that the r-protein genes evolve more slowly than photosynthetic genes. Therefore it would be interesting to analyse other r-protein genes by the same method to know whether we can generalize this observation. Another possibility is that the rpl2 gene represents an exception because of its special localization in the inverted repeat of the chloroplast genome.

Prokaryotic Features Chloroplast and eubacterial ribosomes have several common features. They are similar in size as deduced from sedimentation constants and similar in shape as observed by electron microscopy [25]. Although there are remarkable exceptions which are described later, the primary structure of many chloroplast r-proteins deduced from their genes resemble those of bacteria. The predicted 3D-structure of the spinach chloroplast r-protein L12 by computer-graphic modelling showett that the spatial organization of this protein is largely maintained relative to the 3D-structure of the homologous r-protein from E. coli although at least 40% of the amino acids were found to be different [26]. Common structural features between plastid rproteins of higher plants and E. coli r-proteins have also been suggested by the use of antibodies. Immuno cross reactions have been observed for the following r-proteins: SI, $6, $7, $9, Sll, S12, S19, L1, L2, L3, L6, L13, L17 [27--29]. Immunological similarities between plastid r-proteins from Chlamydomonas reinhardtii and r-proteins from E. coli have also been reported [23,30,31]. Interestingly the chloroplast r-proteins encoded by several nuclear genes also have a prokaryoticlike structure. Their deduced amino acid sequences are in the same range of homology if compared with eubacterial r-proteins as the chloroplast encoded r-proteins (Table I and Ref. 12). Immunological studies have revealed that r-proteins encoded in the plastid genome are structurally more conserved among distantly related slaecies than nuclear encoded r-proteins [23]. This observation is particularly true for the 50S r-proteins and fits with the low evolutionary rate

Table I.

Characteristics of r-proteins encoded in the nucleus and identified by their eDNA.

Species

Name

No. of residues in the Precursor protein

Transit peptide

% of identity homologous part b

No. of residues in terminal extensionsc

Ref.

E. coli-like r-protein Pea Pea Spinach Spinach Spinach Spinach

L9 L24 L 12 L 13 L21 L24

194 194 189 250 256 191

35' 4(P 56 60 55 45 a

33 33 50 56 32 34

10--0 30--21 None 40--9 67--30 23 --23

80 80 82 83 --~ --~

A rabidopsis thaliana

CS 17

154

NK s

39

NKs--20

84

CL18 CL25 L40 • CS-S5

145 104 142 322

50 31 NK ~ 65

NS f NS f N~ NS f

Chloroplast specific r-protein Pea Pea Spinach Spinach

80 80 --~ 85

•Putative number, since the cleavage site of the transit peptide has not been determined. erhe homologous region overlaps almost entirely the E. coli r-protein primary sequence (with the exception of a few amino acid residues in the N or C terminal part). cThe first and second figures correspond to N- and C-terminal extensions to the eubacterial homologous part, respectively. dSubmitted results o f our laboratory. cHomologous to the pea C L - L I 8 protein. fNot significant. ~Not known.

observed for the chloroplast encoded genes compared with nuclear encoded genes [32]. The implication of these observations regarding the advantage of gene transfer from the chloroplast to the nucleus will be considered later. Besides the homology seen between individual E. coli and plastid r-proteins there seems also to exist a similarity in the general architecture of E. coli and chloroplasts ribosomes. In particular, immuno electron microscopic studies of Alaskan pea chloroplast 30S ribosomal subunits have shown that the N6, N6-dimethyladenosine and the 7-methyl guanosine of the 16S rRNA (which are at a position corresponding to A1518-A1519 and to G526 in the E. coli 16S rRNA, respectively) are in an analogous accessible position as in the E. coli 30S subunit [25,33]. All these common structural features naturally deal to the question of whether homologous plastid or bacterial ribosomal components also have

identical function. Several years ago was shown that the E. coli initiation factors IF1, IF2 and IF3 can stimulate the formation o f a translation initiation complex when added to spinach chloroplast ribosomes in the presence of f-Met tRNA and poly(U) [5]. Also, plastid 5S rRNA can replace Bacillus stearothermophilus 5S rRNA in reconstitution experiments and the heterologous 50S subunit still functions in protein synthesis [33]. The most significant demonstration that chloroplast r-proteins may retain their prokaryotic function has been reported recently by Liu et al. [35]. These authors could show that the Chlamydomonas reinhardtii rpsl2 gene is expressed in E. coli cells and that its gene product is assembled into bacterial ribosomes that function efficiently in vivo. Indeed this result is expected since the chloroplast r-protein S12 shares more than 700/0 amino acid identity with its E. coli counterpart. One could speculate that S12 has kept a key func-

tion in the control of initiation and fidelity of translation [36] as also suggested by results obtained from streptomycin resistant mutants. In brief, a single amino acid change responsible for resistance was localized in the rps 12 gene of maternally inherited streptomycin resistant mutants of Chlamydomonas and tobacco [35,37,38]. The mutated plastid S12 r-protein differs in electrophoretic migration from the wild type S 12 protein, a convenient property which was used to characterize the mutants. In E. coli, the same amino acid change in r-protein S12 also results in streptomycin resistance (see Ref. 35 for references). Transformation of E. coli wild type cells with the streptomycin-resistant rpsl2 gene from a C. reinhardtii mutant conferred streptomycin resistance on E. coli [35]. This strongly suggests that the C. reinhardtii S12 when incorporated into E. coli ribosomes can control the translational fidelity. S12 therefore appears to have the same function in C. reinhardtii as in E. coli. Other mutations to streptomycin resistance result from base pair change at conserved sites in the 16S rRNA gene [39,40].

r-Protein Genes Encoded in the Chloroplast Genome of Land Plants: Organization and Expression The complete nucleotide sequence analysis of three chloroplast genomes [9--11,41] has revealed a striking conservation of chloroplast genes during land plant evolution. This observation suffers exceptions in the case of a few protein genes: rpl21, rp122 and rpsl6 genes. The rpl21 gene is encoded in the chloroplast genome of Marchantia polymorpha but not in tobacco and rice. Conversely, the rpsl6 gene is present in the chloroplast genome of higher plants but not in M. polymorpha. Also, in the chloroplast genome o f some Leguminosae [42], the rp122 gene is missing. A tempting hypothesis to explain these differences in the presence of r-protein genes in chloroplast genomes is that a recent transfer of genes from the chloroplast to the nucleus has occurred. In our laboratory we showed that the rpi21 gene, which is chloroplast encoded in Marchantia, is encoded in the nucleus o f spinach (Lagrange et al., submitted).

The organization of chloroplast encoded protein genes exhibits certain general features. Some of the genes are located within the inverted repeat and are consequently duplicated. The number of these duplicated genes depends on the length of the inverted repeat. Thus the largest inverted repeat (76 kb) has been observed in Pelargonium and contains several r-protein genes (5 genes have been identified but 10 genes are probably present) [43] while the inverted repeat of M. polymorpha. is much shorter (10 kb) and does not contain any r-protein genes. In the chloroplast genomes of some Legurninosae which do not contain inverted repeats r-protein genes are not duplicated. Also, the rpl2 gene is in a unique sequence region in Chlamydomonas [44]. This large variation suggests that duplication is not directly related to gene expression or to the regulation of the process of ribosome assembly. Indeed, regulation seems to be translational at least for photosynthetic chloroplast genes [45] and there is evidence that this is also true for the rpl2 gene of C. reinhardtii [46]. Often r-protein genes are clustered in chloroplast genomes and are organized in the same order as in the E. coli S10-, spc- and a-r-protein gene operons [9--11,22,47,48] but this type of organization may not have the same meaning. For instance, genes encoding the S10 and the spc operon regulatory repressor proteins L4 and $8 are missing in the chloroplast genome in contrast to E. coli. This suggests that the expression of the rprotein genes organized in operon-like sequences present in the chloroplast genome may not be regulated by the same feed-back mechanism as observed in E. coli [49]. In spinach, it has been shown that the S10- and spc-like operons are cotranscribed confirming that in chloroplasts each of these structures has lost a characteristic of operon to have a specific transcript [48]. It is interesting that the replication origin of the chloroplast genome of one alga (Chlamydomonas) and of a higher plant (maize) has been identified within a cluster of r-protein genes, i.e., in the rpll6 gene [50,51]. But it is not yet clear whether this location has any functional significance. The presence of introns in the-Chloroplast protein genes, rpsl2, rpsl6 and rpll6, may prove to be a general characteristic of all higher plants [11,52--56], but introns are not found in all r-

protein genes and they do not occur in all plant species. An example is the rpl2 gene which is not interrupted in spinach [16] while it contains an intron in other plants [9,10,21,41]. The persistence of introns in some genes and not in others cannot be explained with certainty at the moment. Now I will describe the features of some particularly interesting chloroplast encoded r-protein genes. First, in higher plants the rpsl2 gene is split into three exons. Exons 2 and 3 of the 3' half of the rpsl2 gene are located in the inverted repeat, i.e., they are present two times. Exon 1 is located very distant in the large single copy region of the plastid genome. In tobacco [55] exon 1 is located 29 kb downstream from the exon 2 in one repeat and 86 kb from the other copy of exon 2 in the other repeat. The same organization was found in the plastid genomes of monocots [9,57] or of M. polymorpha [41]. It was found by several groups that exons 2 and 3 are cotranscribed and cisspliced and exon 1 is trans-spliced to exon 2, i.e., the two separate transcripts from the 5' and the 3'rpsl2 gene are joined together to give a mature rpsl2 mRNA [54,56--58]. The rpsl6 gene is located in the large single copy region of the plastid genome and is surrounded by genes coding for tRNAs and not by other r-protein genes. The rpsl6 gene is transcribed and a 1.3-kb mRNA has been detected in vivo [53,59] which corresponds to the size of the gene. In the mustard [59] a capping experiment has demonstrated that the rpsl6 gene transcript is initiated at a promoter where a '-10' Pribnow box is detected but not the '-35' element which is usually, found in chloroplast promoters. As suggested by Neuhaus et al. [59], the recognition of this unusual promoter would require the existence of either a RNA polymerase which is not E. col# like or of a specific factor. Thus the rpsl6 gene may be regulated in a specific manner at the transcriptional level. The rpsl9 gene in higher plants is either fully included in the large single copy region [9,60] or lies partly in the inverted repeat and partly in the large single copy region o f the chloroplast genome [21,61,62]. In the latter case, two different genes coding for putative proteins homologous to the E. coli S19 are present, each having a 5'-end sequence in common, which is located in the inverted repeat

and a specific 3'-end sequence which is located in the large single copy region. It has been shown by Thomas et al. [61] that in spinach one of these genes is expressed and codes for an identified rprotein, the S19, while the other, the rpsl9' gene with a 3'-end located in the unique sequence region and continuous with the 5'-end of rpsl9, is not transcribed. In contrast to the other chloroplast encoded rprotein genes the rpl22 gene codes for a protein which is larger than its E. coil homologue. At the 5' and 3'-ends it contains additional non-E, colilike sequences surrounding the E. coli-like core. These extensions vary considerably among higher plants [22,48,63]. In spinach the rpl22 gene product has been identified and shown to bind specifically the 5S rRNA. In E. coli the L22 does not have such a function [64]. In Legumes, the rpl22 gene is absent from the chloroplast genome and it has been reported to have been transferred into the nucleus [42]. When the sequence is available it will be interesting to compare the nuclear and the chloroplast encoded rp122 genes to see whether the extensions seen in the chloroplast encoded rpl22 gene with respect to E. coli also exist in the nucleus. A putative rpl23 gene has been located within the inverted repeat of the chloroplast genome and its product has the lowest homology to E. coli among chloroplast proteins. In spinach, this gene is frame-shifted [16]. It is composed of 2 0 R F s overlapping by 8 nucleotide residues. S1 mapping has shown the presence of abundant transcripts with 5'-ends located upstream from each of the two reading frames as well as large transcripts covering the entire gene. Nevertheless, no protein corresponding to the product o f the split rpi23 gene has been detected in plastid ribosomes, suggesting that it represents a pseudogene. Zurawski and Clegg [24] compared the coding sequence of the rpl23 gene in several other higher plants and observed extensive additions and deletions indicating that the chloroplast rpl23 gene is probably not expressed in these plants. The open question is whether in higher plants the 'real' rpi23 gene is present in the nucleus or whether plastid ribosomes do not contain a L23°like protein. The absence of the rpl23 gene in the cyanelle DNA of Cyanophoraparadoxa [65] also raises the question

of the expression of this gene in lower organisms. In the rice plastid genome, in addition to the putative rp123 gene which is located in the inverted repeat a pseudo-rp123 gene (ORF42, downstream of rbcL gene) has been observed [9]. The expression of r-protein genes encoded in the tobacco chloroplast genome has been studied by Northern analysis [12,22,53,54]. It was shown that all 20 r-protein genes are transcribed. The rpsl4 transcripts are the most abundant and the rpi36 transcripts are the least abundant [12]. With the exception of the rps 16 gene all transcripts which have been examined so far are part of polycistronic messengers. Co-transcription of an rprotein gene with other ribosomal protein genes [16,22,48], or with adjacent genes coding for proteins of the photosynthetic apparatus [12,66--68] or with tRNA genes [69,70] has been observed. In spinach, a very long transcript of 15 kb has been identified covering the three operon-like structures (SI0-, spc and a-like operons) [48]. These existing data suggest that there is no specific regulation of r-protein genes expression at the transcriptional level. On the contrary, Northern analysis has revealed in all cases a complex pattern of transcripts which indicates regulation at the post-transcriptional level via processing, splicing or specific degradation. Evidence has been given that posttranscriptional regulation of photosynthetic genes exists [45,71] and it is an open question whether such regulation occurs for chloroplast r-protein genes. It would certainly be interesting to study the expression of the r-protein genes during plant development. Preliminary experiments have shown that in most cases r-protein transcripts accumulate very early in seedling development and that r-protein genes are expressed in a light independent manner [59,69,721.

their lower evolutionary position. For instance, several r-protein genes contain introns in contrast to their homologous counterparts in land plants which are continuous. In Euglena gracilis the rp123, rpsl9, rps3 and rpsl4 genes have 3, 2, 2 and 1 introns, respectively [73,74]. However, in contrast to higher plants, the Euglena rpsl2 gene lack an intron [73]. In C. reinhardtii [35] but not in E. gracilis, the rpsl2 gene is separated from the rps7 gene by 2 sequences coding for thylakoid proteins. Christopher and Hallick [75] have observed a new category of introns in the r-protein encoded genes of E. gracilis and have described them in detail. Altogether, these results indicate that gene rearrangements occurred in different ways in algae and in land plants during the course of evolution. In algae some genes of the translational apparatus are still present in the chloroplast genome while in higher plants they have already been transferred to the nucleus. This is the case for tufA gene coding for the elongation factor EF-Tu which is present in the chloroplast genomes of E. gracilis [76], and probably of Chlamydomonas [77]. This will be the case if the rpl3 gene present in the cyanelle DNA of Cyanophora paradoxa is found in the nucleus of higher plants [78]. E. gracilis is a photosynthetic protist which represent a particular phylum in evolution. This was shown by comparison of the 16S rRNA nucleotide sequences of several organisms [79] and confirmed by the demonstration of the presence of the rpl5 gene in the chloroplast genome of E. gracilis [75]. This gene is included in the E. coli spc operon but it has not been found in any other chloroplast genome of green algae or land plants. The rpl5 gene is also present in the cyanelle DNA of Cyanophora paradoxa (Loffelhardt, pers. commun.), raising the question of the phylogenetic relationship if any of this particular endosymbiont to Euglenoids.

Chloroplast Encoded r-Protein Genes in Algae Nuclear Encoded r-Protein Genes

The organization and expression of r-protein genes in algae show some noticeable differences, which will be briefly described in comparison to higher plants. Algae possess primitive features of plastid gene arrangement probably because of

About two thirds of the chloroplast r-protein genes are encoded in the chloroplast genome. Consequently the other genes are expected to be located in the nucleus. Several genes have been

identified as cDNAs. Some characteristics of their gene products are indicated in Table I. The pea L9 and the spinach L 12 proteins have almost the same length as their homologous E. coli r-proteins [80-82]. On the other hand, the Arabidopsis thaliana CS17, the pea L24 and the spinach L13, L21 and L24 proteins have NH2 and/or COOH terminal extensions in addition to regions which are homologous to E. coli [80,83,84; Lagrange et al., submitted]. Finally, certain chloroplast r-proteins (pea L18 and L25; spinach L40 and CS-SS) have no homology with any E. coli r-proteins. These proteins represent a new class of r-proteins that we have proposed to call 'chloroplast specific proteins' [85]. Their existence show that despite many general similarities between chloroplast and E. coli ribosomes important structural differences have also developed during evolution. It would be very interesting to know if these divergent r-proteins correlate with modified or specific functions of the corresponding ribosomes. An interesting r-protein is CS-S5 in spinach [85]. This protein is present in large amounts and in a free state in the chloroplast stroma. This raises the interesting question of its function. One can speculate that this protein plays a function in the regulation of chloroplast protein synthesis. As already mentioned post-transcriptional mechanisms of regulation in chloroplasts are generally recognized [45]. Such regulatory mechanisms have also been proposed by Liu et al. [86] to explain their observation that a preferential synthesis of chloroplast r-proteins occurs in Chlamydomonas mutants which have a low chloroplast protein synthesizing activity. Up to now the mechanisms underlying such types of regulation have not been described at the molecular level. Therefore the CS$5 may represent a tool to examine one type of post-transcriptional regulation in chloroplasts. All of the precursors of the nuclear encoded chloroplast r-proteins have a transit peptide. The maturation of the C. reinhardtii L18 r-protein (equivalent to the E. coli L27) has been reported to be a two step process [87,88]. The second step occurs on the assembled ribosome particle and requires chloroplast protein synthesis [87]. It would certainly be interesting to know the

sequence of the transit peptide of this protein and to identify the two maturation sites.

Coordinated Synthesis of Ribosomal Components and Assembly of Ribosomes With the exception of the L12 protein which is present in several copies per chloroplast ribosome [81] all other r-proteins are present in a stoichiometric amount. We have to ask how coordination of expression of all ribosome constituents is achieved and how ribosome assembly takes place. Only a few publications are available which deal with these two problems. These will be"reviewed now. Feierabend [89] found that elevated temperature blocks the formation of plastid ribosomes in higher plants but chloroplast proteins synthesized in the cytoplasm are still accumulated [90]. This suggests that there is a pool of unassembled r-proteins. A marked accumulation of 7 r-proteins which should be of cytoplasmic origin was observed [90]. It would be interesting to know whether these proteins are still synthesized at normal temperature. This would give information whether the synthesis of proteins in the two genetic compartments is coordinated or not. Complete assembly of ribosomal subunits has been detected in isolated chloroplasts which indicates that either pools of free nuclear coded r-proteins exist in the organelle, or that individual rproteins are being released from existing ribosomal subunits and reutilised [14]. The pool sizes of unassembled r-proteins are presumably small in comparison to the two roproteins present in a free state in large amounts in the stroma [85]. The presence of ribosomal particles smaller than 30S or 50S subunits has also been detected in chloroplasts suggesting at least a two-step ribosome assembly [14]. The assembly of the E. coli ribosomal subunits has been studied in detail by reconstitution techniques. It has been found that only seven of the 30S r-proteins bind individually to 16S rRNA (see Ref. 91 for references). Using proteins blotted on a nitrocellulose filter Rozier and Mache [92] found that a set of 7 spinach chloroplast r-proteins are able to bind to chloroplast or to E. coli 165 rRNA.

These 7 proteins could participate to the early assembly of the 30S subunit. Interestingly, 4 of these 7 proteins are synthesized within the chloroplast. It would not be surprising to find that the proteins coded by the chloroplast rps4 and the rps7 genes are included in this set of 16S rRNA binding proteins since in E. coli, $4 and $7 initiate the assembly process of the 30S subunit [91]. Using the same method of detection of rRNA binding proteins, Toukifimpa et al. [64] found that the 5S rRNA binds to 2 chloroplast r-proteins of the large subunit instead of 3 as in E. coli (L5, L18 and L25). These results were confirmed by foot-printing experiments. Surprisingly, one of the chloroplast 5S rRNA binding proteins is homologous to the E. coli L22 which is probably located close to the channel for mRNA entry [93]. A detailed study of the binding sites of the 2 chloroplast r-proteins has shown that several structural differences in the 5S rRNA-protein complex exist between the chloroplast and E. coli [64]. Why are some Genes of Ribosomal Proteins Still Present in the Chloroplast genome?

Chloroplast evolution has been marked by the transfer of genes to the nucleus. In the land plant Marchantia and in higher plants the number of rprotein genes encoded by the chloroplast genome is the same as are most of the genes themselves. This suggests that the transfer process has stopped or is very rare. Why? Several hypotheses can be postulated to answer this question: (1) The genetic exchange took place under conditions which are now lacking. (2) The r-proteins encoded by the plastid genome are constituents of a core particle. The assembly of this core particle requires the presence of a limited number of proteins synthesized coordinately with the rRNA species. The chloroplast Would keep the precise control of the synthesis of these proteins. The 4 r-proteins which have been found to bind to the 16S rRNA and are encoded in the chloroplast [92] support this hypothesis and so does the recent finding that the chloroplast encoded rpl22 gene product binds to 5S rRNA [64]. Thus the genes whose protein products strongly interact with rRNA would not have escaped from the chloroplast and the conservation

of the r-protein genes in the plastid genome would coincide with the early assembly sequence. (3) One can also speculate that certain r-protein genes are retained in the chloroplast because they can only tolerate new mutations to a very limited extent. The chloroplast genome which has a low evolutionary rate [32] would be more advantageous to these genes than the nuclear genome. The corresponding proteins would have a specific function which was elaborated a very long time ago. For example, the L2 and L16 r-proteins are involved in the E. coli peptidyl transferase center [94] and their chloroplast counterparts, which are chloroplast encoded, might have kept the same function. Indeed we do not really know how the cell benefits from having nuclear encoded r-protein genes. Is it only for controlling the development of plastids? This review of the main results obtained concerning chloroplast ribosomal proteins shows that there are many problems still to be solved. Problems of gene transfer from the chloroplast genome to the nucleus, of specific structural features in relation with the evolution, problems of regulation of gene expression, of coordinated assembly. All these questions show the fundamental interest of studies concerning chloroplast ribosomes. References 1 2 3

4

5

6

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