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Home , Colicin, Conserved sequence, Cuscuta europaea, Leucoplast, Polytoma

MICROBIOLOGICAL REVIEWS, Dec. 1994, p. 700-754 Vol. 58, No. 4 0146-0749/94/$04.00+0 Copyright © 1994, American Society for Microbiology and Synthesis ELIZABETH H. HARRIS,`* JOHN E. BOYNTON,' AND NICHOLAS W. GILLHAM2 DCMB Group, Departments of Botany' and Zoology,2 Duke University, Durham, North Carolina 27708-1000

ORIGIN OF ...... 700 CHLOROPLAST STRUCTURE AND CONTENT.. ..700 THE PROCESS OF CHLOROPLAST PROTEIN SYNTHESIS...... 702 'rnl Initiation...... t.a..i.....n...... 1.... F' kElongation-s - ...... 17d 03 .4A04 Chloroplast tRNAs and Aminoacyl-tRNA Synthetases...... 704 FOR rRNAs ...... 704 Phylogenetic Conservation...... 704 General Characteristics of Chloroplast rRNA Gene Organization...... *707 16S rRNA ...... 707 23S rRNA...... 709 5S rRNA...... o..709 in rRNA Genes ...... 709 The 16S-23SSpacer...... 712 tRNAs Flanking the rRNA ...... 712 Antibiotic Resistance Mutations in the Chloroplast rRNA Genes...... 1.....2.0...... 712 RIBOSOMAL ...... 714 Number and Nomenclature ...... 714 Organization of Chloroplast Genes ...... *...... 715 Correspondence of Chloroplast Ribosomal Proteins to Bacterial Ribosomal Proteins...... 716 Proteins of the Small Subunit...... 716 Proteins of the Large Subunit...... 726 Chloroplast Ribosomal Proteins with No Obvious to Those of E. coli ...... 729 Comparative Analysis of Ribosomal Proteins...... 30 ASSEMBLY OF CHLOROPLAST RIBOSOMES ...... 730 SYNTHESIS OF THE COMPONENTS OF CHLOROPLAST RIBOSOMES ...... , .. 731 of rRNA Genes...... 731 Transcription of Chloroplast Genes Encoding Ribosomal Proteins ...... 732 Posttranscriptional Regulatory Mechanisms Affecting Chloroplast mRNAs...... 733 Membrane Binding of Chloroplast Ribosomes ...... 733 HOW ESSENTIAL IS CHLOROPLAST PROTEIN SYNTHESIS?...... -...... '...... 734 CONCLUSIONS ...... 735

ACKNOWLEDGMENTS...... 735 REFERENCES ...... I...... "o ..73

ORIGIN OF CHLOROPLASTS among these various taxa have produced intriguing directions for future evolutionary studies, while analysis of ribosomal Chloroplasts and mitochondria contain protein synthesizing- protein sequences, particularly among the diverse algal groups, systems more similar to those of than to those of the promises. to be a valuable tool for determining conserved eukaryotic , consistent with the hypothesis that these regions likely to have essential functions in assembly had xenogenous (endosymbiotic) rather than autog- or protein synthesis. enous (intracellular differentiation) origins (see. references 5, 205, 220-223, 274, 633, and 694 for discussions). Phylogenies CHLOROPLAST GENOME STRUCTURE AND based mostly on rRNA sequences indicate that the cyanobac- GENE CONTENT teria are ancestral to chloroplasts while the members of the alpha subdivision of the purple sulfur bacteria are the likely Unlike their prokaryotic ancestors, neither chloroplasts nor progenitors of mitochondria (221, 222). Whether the, chloro- mitochondria are genetically autonomous, and information phyte algae and land on the one hand, and the rhodo- specifying components of the protein synthesizing phyte, chromophyte, and euglenoid algae on the other repre- systems is divided between organelle and nucleus. Separation sent more than one endosymbiotic event remains unresolved of the genes encoding these and proteins between two (130, 403, 434). Comparisons of gene order and arrangement discrete cellular compartments suggests that mechanisms must have evolved to coordinate expression of these genes so that protein synthesis in the organelle can proceed efficiently. * Corresponding author. Mailing address: DCMB, Duke University Whereas chloroplast of land plants usually have a Box 91000, Durham, NC 27708-1000. Phone: (919) 613-8164. Fax: common organization and gene content, a great deal more (919) 613-8177. Electronic mail address: [email protected]. variability is encountered among the algae, particularly with 700 VOL. 58, 1994 CHLOROPLAST RIBOSOMES AND PROTEIN SYNTHESIS 701

Fabaceae (314, 482, 487), which have lost the inverted repeat and thus contain only a single copy of each of the rRNA genes. Black pine (Pinus thunbergii) chloroplast DNA does possess a short inverted repeat sequence, which contains a tRNA gene and part of the 3' portion of the psbA gene, but not the rRNA genes (654). In contrast, species with the largest chloroplast genomes often have expanded inverted repeats (e.g., Pelargo- nium hortorum has a 76-kb inverted repeat encompassing nearly half of the 216-kb chloroplast genome, in which many genes normally in the single-copy region have been duplicated [482]). Chloroplast genomes from land plants specify a relatively constant set of components for the protein-synthesizing ma- chinery of the organelle (4 rRNAs, 30 to 31 tRNAs, 21 ribosomal proteins, and 4 RNA polymerase subunits) and for photosynthesis (28 thylakoid proteins plus 1 soluble protein, the ribulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco] large subunit). In addition, homologs of 11 subunits of mam- malian mitochondrial complex I (the ndh genes) have now been found to be encoded by chloroplast DNA in flowering plants and Marchantia species (9,713). Chloroplast genomes of FIG. 1. Schematic diagram of a typical land chloroplast genome (tobacco), showing the positions of the inverted repeat, rRNA gymnosperms, liverworts, and algae (e.g., genes, and genes encoding ribosomal proteins. Gene locations are reinhardtii) which synthesize chlorophyll in darkness possess from reference 560. genes encoding three subunits of a light-independent proto- chlorophyllide reductase that is also found in photosynthetic prokaryotes (see reference 367 for a summary). These genes are absent from the tobacco and rice chloroplast genomes. regard to the ribosomal protein genes that have been retained Mapping and sequencing studies of chloroplast genomes in the organelle. In this section we review the chloroplast from widely different algal taxa reveal that these are much genome structure of land plants and the algal genera that have more variable in organization and gene content than those of been investigated to date with respect to composition and land plants. The well-characterized chloroplast genomes of organization of genes encoding rRNAs and ribosomal pro- three species of unicellular green algae in the genus Chlamyd- teins. omonas are substantially larger (C. reinhardtii, 196 kb; C. Chloroplasts are highly polyploid organelles containing cir- eugametos, 243 kb; C. moewusii, 292 kb) than the chloroplast cular DNA molecules of 85 to 200 kb organized into discrete genomes of land plants (42, 43, 50, 247). In these species the membrane-associated (see references 50, 206, 260, two copies of the large inverted repeat encoding the rRNAs 337, 482, 483, 558, 613, and 614 for reviews). Three land plant are separated by unique sequence regions of roughly equal chloroplast genomes have been completely sequenced: the size. Chloroplast genes in Chlamydomonas species are also dicotyledon tobacco (Nicotiana tabacum, 156 kb [560, 561]), extensively rearranged between distantly related species and the monocotyledon rice (Oryza sativa, 135 kb [265]); and a with respect to land plants (43). The green alga Spirogyra liverwort (Marchantia polymorpha, 121 kb [471-473]). Each maxima, in the charophyte presumed to be ancestral to contains 110 to 120 genes (482, 614). These sequences, as well land plants, lacks an inverted repeat and shows altered gene as restriction maps and partial sequences from many other order relative to land plants (352, 393). species, indicate that the basic chloroplast genome structure The organization, structure, and gene content of the com- and overall gene order in land plants are highly conserved. pletely sequenced 145-kb chloroplast genome of Euglena gra- Although green algae () are regarded as ancestral cilis Z (243) depart markedly from the chloroplast genomes of to land plants, modern green algae often show substantial chlorophyte algae or land plants. In this Euglena strain and in rearrangements in chloroplast gene order (see below). Other its colorless relative Astasia longa, the plastid genome contains groups of algae (Rhodophyta, Euglenophyta, Chromophyta) three tandemly repeated rRNA operons plus an additional 16S show even more diversity in gene content and organization. gene or fragment thereof (288, 289, 315-317, 478, 569). In the typical land plant chloroplast genome, unique se- Euglena gracilis var. bacillaris has only a single complete rRNA quence regions of 15 to 25 kb and 80 to 100 kb are separated (720). Most of the Euglena chloroplast tRNA genes are by the two copies of an inverted repeat, which is usually 20 to grouped in tight clusters of two to five genes, whereas they tend 30 kb in size and contains genes encoding the chloroplast to be scattered in plastid genomes of land plants. While most rRNAs, certain tRNAs, and often one or more genes specify- protein-coding chloroplast genes in land plants or Chlamydo- ing proteins (Fig. 1) (see references 482 and 614 for reviews). monas species are uninterrupted or contain at most one or two Within the inverted repeat, the rRNA operon is usually introns, comparable genes in Euglena gracilis each contain oriented with the 23S rRNA gene closer to the small single- multiple introns (243, 482). However, several chloroplast copy region and the 16S rRNA gene closer to the large tRNA genes that have introns in land plants lack introns in single-copy region. The two repeats are identical in sequence Euglena or Chlamydomonas species (331). A number of other as a consequence of an active copy correction system (50). genes found in land plant chloroplast genomes, including three Nearly two-thirds of the variation in size among land plant genes encoding ribosomal proteins, are missing from the chloroplast genomes (120 to 216 kb) is accounted for by Euglena chloroplast genome (see below), but this algal genome expansion or contraction of the inverted repeat (482). The also contains some genes not found in plastid genomes of land smallest chloroplast genomes among land plants are seen in plants. conifers (355, 600, 695) and in six tribes of the legume family The plastid of Cyanophora paradoxa is often referred to as 702 HARRIS ET AL. MICROBIOL. REV. the cyanelle because of its secondary peptidoglycan wall and chloroplast protein synthesis has been presented by Steinmetz photosynthetic apparatus with phycobiliproteins typical of cya- and Weil (593). nobacteria and red algae. Most of the 133-kb cyanelle genome has now been sequenced (32, 598). This genome contains an Initiation inverted repeat which encodes the cyanelle rRNAs and several other genes. Although the gene content of the cyanelle gener- In prokaryotes, protein synthesis begins with formation of a ally resembles that of land plant chloroplasts, there are about preinitiation complex from the 30S ribosomal subunit and 30% more genes, including 11 additional genes encoding tRNAIMetUAC, with the 30S subunit binding to the purine-rich components of the translational apparatus. So far, only a single Shine-Dalgarno sequence 7 ± 2 nucleotides (nt) upstream of type I has been found in Cyanophora paradoxa, in a the initiator AUG (230, 261, 323). The canonical Shine- tRNAIeU gene (162). The same intron is found in cyanobacte- Dalgarno sequence, GGAGG, or a variant, pairs with a ria. pyrimidine-rich complementary sequence, the anti-Shine-Dal- algae contain a plastid and residual nucleus or garno sequence, near the 3' end of the 16S rRNA molecule. nucleomorph enclosed within the of the Addition of a50S ribosomal subunit converts the preinitiation cytoplasm and thus effectively separated from the normal complex to an initiation complex that can enter the elongation nucleus (see 123, 386). Distinctly different 18S rRNAs are phase of protein synthesis. These reactions are promoted by encoded by the nucleus and nucleomorph of (D the three initiation factors, IF-1, IF-2, and IF-3. IF-1 enhances and are spatially separated within the cell (129, 414). The the rates of ribosome dissociation and association and the nucleomorph rRNA genes are related to those of red algae, activities of the other initiation factors (261). IF-2 is involved in while the nuclear rRNA genes are clustered separately in the initiator tRNA binding and GTP hydrolysis, while IF-3 pre- phylogenetic branch containing land plants and green algae. vents ribosomal subunit association in the absence of mRNA This suggests that cryptomonad algae may have arisen through and appears to stabilize mRNA binding by promoting the a second endosymbiotic event in which a eukaryotic symbiont conversion of a preternary ribosome-mRNA-fMet tRNA com- from the red algal lineage was taken up by a unicellular host plex into a ternary complex in which codon-anticodon interac- more closely related to the green algae (129, 130, 386). Partial tion has occurred. IF-3 also is thought to proofread the sequencing of the plastid genome of Cryptomonas (D has AUG-anticodon interaction. Chloroplast equivalents of IF-2 revealed the presence of several novel genes, including four and IF-3, designated IF-2Ch, and IF-3Chl, have been character- genes for ribosomal proteins not found in chloroplast genomes ized from Euglena gracilis (212, 324, 325,375,527, 678). Roney of land plants (122, 124, 680; also see below). et al. 527) confirmed that IF-2Chl is required for binding of In the red alga Porphyra purpurea, over 125 genes have been tRNA et to chloroplast 30S subunits, as is prokaryotic IF-2. identified in the ca. 60% sequenced chloroplast genome (514, IF-2,hl occurs in several complex forms, varying in molecular 515), suggesting that the entire genome may contain as many mass from 200 to 800 kDa (375). Subunits of 97 to >200 kDa as 200 to 220 genes, about twice as many as found in the have been observed in these preparations. IF-3,hl promotes completely sequenced genomes of land plant chloroplasts. initiation complex formation in the presence of IF-2Chi. Al- These include at least seven photosynthesis and nine ribosomal though IF-3Chl will replace IF-3 in initiation protein genes not present in land plants. Introns have not been complex formation, there is some evidence that its function found in any of the 80 genes sequenced to date. The chloro- may be modified (527). plast genome of P. yezoensis possesses an inverted repeat A DNA sequence with homology to the E. coli infA gene containing the rRNA genes (353, 562, 563), but the related red encoding initiation factor IF-1 has been identified in the algae P. purpurea and Grijflthsia pacifica lack this inverted- chloroplast genomes of land plants, including the colorless repeat structure. In P. purpurea the rRNA genes are encoded parasite Epifagus virginiana (558, 714), but is apparently absent in direct repeats which are not identical in sequence (514, 516, from the completely sequenced chloroplast genome of Euglena 563). The unicellular red alga Cyanidium caldarium possesses gracilis (243). The tobacco infA gene, in contrast to the spinach an inverted repeat containing only the rRNA genes, but gene gene (571), lacks the ATG initiation codon and order appears to be more similar overall to that of Cryptomo- thus may be a pseudogene. Reading frames with homology to nas (D than to that of P. yezoensis or Griffithsia pacifica (385). the genes encoding IF-2 and IF-3 have not been detected in the Inverted repeats containing rRNA genes are also found in sequenced plastid genomes of green plants, Epifagus virginiana, the plastid genomes of the brown alga Dictyota dichotoma or Euglena gracilis, and inhibitor experiments suggest that the (330) and the golden-brown algae Olisthodiscus luteus and Euglena genes specifying these factors are nuclear in location. Ochromonas danica (108, 563). The plastid genome of the However, homologs of the infB gene encoding IF-2 have been brown alga Pylaiella littoralis contains two different circular found in the chloroplast genomes of the red algae P. purpurea DNA molecules (369, 370, 404, 405). The larger (133 kb) (514) and Galdieria sulphuraria (322). Lin et al. (359) have molecule resembles a typical land plant chloroplast genome, recently reported characterization of a cDNA clone encoding with two rRNA operons in an inverted repeat. The smaller (58 IF-3Chl in Euglena gracilis. This nuclear gene appears to be kb) molecule contains a 16S pseudogene sequence, which is present in about four copies, one of which is probably a 65% homologous to the functional 16S genes of the large pseudogene. The putative protein contains two acidic regions molecule, and a complex region that hybridizes with a 23S with no homology to other known sequences, in addition to a rRNA probe (369, 370). 175-amino-acid region with 31 to 37% homology to other IF-3 proteins. THE PROCESS OF CHLOROPLAST Shine-Dalgarno-like sequences are present in the untrans- PROTEIN SYNTHESIS lated leader regions of many but not all chloroplast mRNAs (35,44, 318, 532, 593, 746). Ruf and Kossel (532) reported that We begin this brief review of chloroplast protein synthesis 37 of 41 chloroplast genes examined in tobacco have such with a comparison with the process as it occurs in bacteria. This sequences if one extends the anti-Shine-Dalgarno sequence in section will be followed by discussion of the tRNAs and the 16S rRNA beyond the canonical CCUCC to include the aminoacyl-tRNA synthetases. A more detailed discussion of adjacent unpaired ACUAG sequence. Bonham-Smith and VOL. 58, 1994 CHLOROPLAST RIBOSOMES AND PROTEIN SYNTHESIS 703

Bourque (35) observed that 181 of 196 chloroplast-encoded tRNA. The specific ternary complex is selected on the basis of transcripts examined possessed a Shine-Dalgarno sequence codon-anticodon recognition at the A site and is followed by within 100 bp 5' to the initiation codon. However, spacing of GTP hydrolysis and the release of an EF-Tu-GDP complex. Shine-Dalgarno sequences in chloroplast mRNAs is less uni- Peptide bond formation takes place with transfer of the form than in bacteria. Frequency distributions of the most growing peptide chain to the aminoacyl-tRNA in the A site. common individual positions potentially involved in - Translocation is promoted by EF-G and GTP hydrolysis, and ing with 16S rRNA ranged from -2 to -29, with a major peak involves movement of the peptidyl-tRNA-mRNA complex (ca. 40%) at -7 to -8, a smaller peak at -15 to -16, and a from the A to the P site. The process is then repeated, and the third small peak at -21 to -23 (35, 532). Thus, chloroplast deacylated tRNA moves from the P to the E site. The A and E ribosomes may be able to accommodate larger distances sites themselves are allosterically linked in a negative sense so between the ribosome recognition site and translational start that occupation of the A site by aminoacylated tRNA reduces sites than bacterial ribosomes. For example, in the Chlamydo- the affinity of the E site for deacylated tRNA and vice versa. monas rpsl2 gene, a canonical Shine-Dalgarno sequence is Regeneration of the active EF-Tu-GTP complex from EF-Tu- found at position -55 upstream of the initiator codon (364). GDP is mediated by elongation factor EF-Ts. All three elon- The variability of the Shine-Dalgarno sequence raises the gation factors have been characterized from Euglena chloro- question whether initiation from this sequence proceeds as in plasts by Spremulli and colleagues (53, 145, 173, 341, 585), and eubacteria for Shine-Dalgarno sequences close to the AUG the structure of the guanine nucleotide-binding of codon and occurs by transient binding and "scanning" for EF-Tu has been modeled by Lapadat et al. (341). EF-Tu has more-distant Shine-Dalgarno sequences (306). In tobacco the also been purified from pea and tobacco chloroplasts (445, mRNAs for those chloroplast genes lacking Shine-Dalgarno 589). sequences either show only a trinucleotide sequence for po- Reading frames with homology to the bacterial genes en- tential base pairing (atpB) or contain out-of-frame initiator coding the three elongation factors EF-Tu, EF-G, and EF-Ts codons between the potential recognition sites and the respec- are absent from the three completely sequenced land plant tive in-frame start codons (rpsl6, rpoB, and petD [532]). chloroplast genomes (482, 558), but some of these genes have In Euglena chloroplasts, mRNA-rRNA recognition seems to been retained in the plastid genomes of certain algae (see proceed by somewhat different rules, because the putative below). Two distinct nuclear genes encoding chloroplast anti-Shine-Dalgarno sequence CUCCC differs from the canon- EF-Tu have been identified in tobacco (445, 611, 661). A ical CCUCC sequence and actually forms the 3' terminus of nuclear EF-G gene has been cloned and sequenced from the 16S rRNA rather than being located several bases from the soybean (650), and a partial clone obtained from pea (2). end (592). Since only about half of the Euglena chloroplast Early inhibitor experiments with Euglena gracilis indicated mRNAs contain Shine-Dalgarno sequences, two modes of that EF-Ts and EF-G were nuclear gene products but that initiation complex formation have been postulated (527, 677). EF-Tu might be encoded in the chloroplast (52, 173). These In one class of mRNAs, complex formation is facilitated by a predictions were confirmed by identification of a chloroplast Shine-Dalgarno-like sequence. However, in the second class tufA gene encoding EF-Tu (429) and by failure to find genes the A+U content of the region 5' to the initiator AUG is 90% encoding EF-Ts or EF-G in the recently completed Euglena or greater and this portion of the mRNA is relatively unstruc- chloroplast genome sequence (243). The Euglena tufA gene is tured, making potential start sites in this region readily acces- split into three separated by two introns (429). An sible to small subunits. Koo and Spremulli (318, 319) have uninterrupted sequence with homology to theE. coli tufA gene studied formation of initiation complexes in vitro with tran- has been reported from the chloroplast genome of C. rein- scripts containing the 5' untranslated leader region of the hardtii (15, 684). The tufA gene sequence is also found in the Euglena rbcL mRNA, which is A+U rich and contains no Cyanophora cyanelle genome (32, 598) and in the chloroplast Shine-Dalgarno sequence. Introducing a Shine-Dalgarno se- genomes of representative green algae in the families Ulvo- quence into this region enhanced initiation only slightly. phyceae, , and Charophyceae, the latter group and/or modification of the leader region demon- being the presumed ancestors of land plants (14, 15). However, strated that a minimum of about 20 nt is required to form the tufA is absent from the chloroplast genome of the liverwort initiation complex in vitro and that the full 55-nt length is Marchantia polymorpha, representative of the earlier land plant necessary for full activity in complex formation (318). The lineages (472, 473). Baldauf and Palmer (15) concluded that primary sequence of the region seems less important for transfer of this gene to the nucleus probably occurred in the initiation than does its length. The native 55-nt sequence has charophycean lineage prior to the emergence of land plants. only weak secondary structure, and modification of the se- Reith and Munholland (514) have reported that the chloro- quence to create increased secondary structure within about 10 plast genome of the red alga P. purpurea not only possesses a nt of the AUG codon diminished formation of the initiation reading frame corresponding to tufA but also possesses one complex significantly (319). Koo and Spremulli concluded that corresponding to tsfwhich encodes EF-Ts in prokaryotes. This the major determinant of initiation in those Euglena mRNAs gene has also been found in the chloroplast genome of the with no Shine-Dalgarno sequence is presence of the AUG thermophilic red alga Galdieria sulphuraria (322). In pro- codon in an unstructured region of mRNA that is accessible to karyotes, mutations to fusidic acid resistance can occur in the the 30S subunit. structural gene for EF-G (fus) (357). A nuclear mutation in C. reinhardtii has been reported to confer fusidic acid resistance Elongation on chloroplast EF-G, but the gene encoding this factor has not yet been identified (74; also see 247). Elongation of the peptide chain requires three steps, i.e., Production of chloroplast protein synthesis factors appears aminoacyl-tRNA binding, peptide bond formation, and trans- to be light regulated. Spremulli and coworkers have shown that location, and involves three binding sites for tRNA (261, 460, activities of Euglena IF-2, IF-3, EF-Tu, EF-G, and EF-Ts all 518, 690). The aminoacylated tRNA combines with elongation increase on transfer of cells from dark growth to light (52, 173, factor EF-Tu and GTP to form a ternary complex, which then 324, 585). In Chlamydomonas synchronous cultures, transcrip- associates with a ribosome complexed to mRNA and peptidyl- tion rates for four chloroplast-encoded photosynthetic genes 704 HARRIS ET AL. MICROBIOL. REV. and for the tufA gene were all found to be maximal at the Several chloroplast tRNAs have unusual features. TIwo beginning of the light period (51, 350). However, EF-Tu different tRNAIle species are found in plant chloroplasts. The mRNA decreased to almost undetectable levels in the second major species (tRNAIle1, encoded in the spacer between the half of the light period. Activity of the pea chloroplast EF-G, 16S and 23S genes) recognizes the codons AUU and AUC, encoded by a nuclear gene, is also light regulated but at the while a minor species (tRNAIle2) recognizes AUA. However, level of translation (1). the gene encoding the latter tRNA contains a CAU anticodon, which normally would recognize AUG for methionine. One Termination possible explanation is that the C residue is modified in some way posttranscriptionally. In E. coli the C of the homologous Termination of translation in bacteria involves the hydrolysis tRNA is modified to lysidine, a novel type of cytidine with a of peptidyl-tRNA and release of the completed protein from lysine residue, which allows it to recognize the AUA codon the ribosome when the ribosome reaches one of the three (444). termination codons (261). Termination requires the action of One tRNAGlUuc has a special function in chlorophyll two release factors, RF-1, which is specific for UAA and UAG, biosynthesis as well as participating in protein synthesis, while and RF-2, which is specific for UAA and UGA. A third release the other two species have a U*UG anticodon specific for factor, RF-3, stimulates the activities of RF-1 and RF-2. The glutamine and are converted from Glu-tRNA01n to Gln- same three codons are used for translation termination in tRNAGJn by a specific amidotransferase activity present in chloroplasts (35, 36), with UAA being by far the most frequent chloroplast extracts (398, 616). This mischarging mechanism (70% in land plant sequences surveyed by Bonham-Smith and has also been described in several gram-positive bacteria (398). Bourque [36]) and UGA being rare (9%). UAA is also The chloroplast genomes sequenced to date encode a typical overwhelmingly preferred as the stop codon in Chlamydomo- initiator tRNA"cICAu, and all employ the three classical nas chloroplast genes (247). Bonham-Smith and Bourque (35) termination codons (UAA, UAG, and UGA). However, genes noted that UGA was never used as a stop codon in Marchantia for tRNAs recognizing the codons CUU/C (Leu), CCU/C chloroplast genes and proposed that a modification of the 16S (Pro), GCU/C (Ala), and CGC/A/G (Arg) are absent from the rRNA in this species prevents recognition of UGA as a chloroplast genomes of tobacco and rice. Since all 61 sense termination signal. No reading frame with homology to any of codons are used in the three sequenced land plant chloroplast the genes encoding bacterial termination factors has been genomes, this deficit in specific tRNAs requires that the identified in a chloroplast genome, nor has isolation of these tRNAs either be imported or be read by the "two-of-three" factors been reported. mechanism used in mitochondria (174, 716) or by four-way wobble (480). In the absence of import in Euglena Chloroplast tRNAs and Aminoacyl-tRNA Synthetases chloroplasts, one of the last two mechanisms would have to pertain to seven of the eight codon families (243). In land plant The properties of chloroplast aminoacyl-tRNA synthetases chloroplasts, two-of-three or four-way wobble seems to be used have been summarized by Steinmetz and Weil (593). These for tRNAMIaUda,GC, tRNAProU*GC, and tRNAA`gICG, which can enzymes are encoded in the nucleus. Most are distinguishable read respectively all four alanine (GCN), proline (CCN), and from their cytoplasmic counterparts and will charge only arginine (CGN) codons (488). The first two tRNAs contain a chloroplast or prokaryotic tRNAs efficiently. These enzymes modified U (U*) in the anticodon. The problem of decoding have unusually high molecular masses (75 kDa or greater) and the six leucine codons is solved somewhat differently. Two of can be found as monomers, homodimers, heterodimers, or the leucyl-tRNAs translate the UUA and UUG codons (488). heterotetramers depending on the enzyme. The remaining tRNAeUUAM7G translates all four CUN The structure and codon recognition patterns of chloroplast codons for leucine apparently by a U * N wobble mechanism tRNAs and the organization of their cognate genes have been (489). extensively reviewed elsewhere (397-399, 593, 616). Genes In tobacco, rice, and liverwort, six of the chloroplast-en- encoding individual chloroplast tRNAs are highly conserved in coded tRNA genes possess introns which must be removed different species of land plants and are similar in structure and from the primary transcript during processing (398). In to- sequence (ca. 70% sequence identity) to prokaryotic tRNA bacco these introns range in size from 503 bp (tRNAeuuAA) genes but have low homology to those of eukaryotic cells. to 2,526 bp (tRNALYSJuu) (616). Many land plant chloroplast However, the 3'-terminal CCA triplets of chloroplast tRNAs tRNAs are singly transcribed, although a cotranscribed, tricis- are added posttranscriptionally, as occurs for all eukaryotic tronic tRNA gene cluster has been identified in tobacco (398) cytoplasmic tRNAs but for only about one-third of bacterial and the two tRNAs found in the spacer between the 16S and tRNAs. Isoaccepting tRNAs for a given are 23S rRNA genes are transcribed as part of the rRNA operon encoded by different chloroplast genes, but these tRNAs are precursor (see below). Cotranscription of tRNA gene operons charged by the same chloroplast tRNA synthetases. Some is the usual case in Euglena gracilis. RNase activities thought to chloroplast tRNA genes are preceded by prokaxyotic-like be involved specifically in tRNA processing have been identi- sequences, but such sequences are absent upstream fied in chloroplast extracts (225, 226, 727). of other chloroplast tRNA genes, which may thus possess alternative promoters, possibly internal to the (227, 229, 616). PLASTID GENES FOR rRNAs The tobacco chloroplast genome contains 30 tRNA genes, 23 of which are single and 7 of which are duplicated in the Phylogenetic Conservation inverted repeat. Rice has the same set of tRNA genes as All chloroplast genomes examined contain genes for the tobacco, but the inverted repeat extends through the tRNAH1S 16S, 23S, and 5S RNAs of the chloroplast ribosome. Table 1 gene, found in the single-copy region adjacent to the inverted lists species for which sequences have been published. Chlo- repeat in tobacco. In liverwort there are 31 chloroplast- roplast rRNAs are highly conserved at the sequence level and encoded tRNA genes, with the extra gene being tRNAAxgCCG, are most closely related to eubacterial sequences, which in- but in Euglena gracilis there are only 27 (243, 558, 616). clude those of (210, 219, 236, 512, 709). For VOL. 58, 1994 CHLOROPLAST RIBOSOMES AND PROTEIN SYNTHESIS 705

TABLE 1. Plastid and cyanobacterial rRNA sequences published or submitted to GenBank Taxon GenBank accession no(s). Reference(s) 16S rRNA Anabaena sp. X59559 356 Anacystis nidulans X03538; X00346, K01983 (partial) 333, 647, 697 Antithamnion sp. X54299 384 Astasia longa X14386 569 Chlamydomonas moewusii X15850 140 Chlamydomonas reinhardtii J01395, X03269 137 Chlorella ellipsoidea X12742, X05694, X03848 722, 724, 725 Chlorella kessleri X65099 281 Chlorella mirabilis X65100 281 Chlorella protothecoides X65688 280 Chlorella sorokiniana X65689 280 Chlorella vulgaris X16579 279 Conopholis americana X58864 702, 703 Cryptomonas 1 X56806 130 Cyanidium caldarium X52985 383 Cyanobacteria (miscellaneous spp.) M63813, M63814 63 M62775, M62776 681-683, 692 M64522, M64526, M64531, M64536 548 Cyanophora paradoxa M19493 (partial) 287 Daucus carota X73670 395 Epifagus virginiana M81884, X62099 435, 712, 714 Euglena gracilis V00159, X12890, X05005, X70810 217, 243, 529, 530, 549 Euglena gracilis bacillaris X00536 (partial) 152 Glycine max X07675, X06428, M37149 (partial) 110, 673 Helianthus annuus X73893 77 Marchantia polymorpha X04465 312, 472, 473 Nanochlorum eucaryotum X76084 553 Nicotiana plumbaginifolia M82900, X70938 476, 729 Nicotiana tabacum J01452, J01453, V00165, V00166, Z00044 560, 561, 645, 646 Ochromonas danica X53183 705 Ochrosphaera sp. X65101 281 Olisthodiscus luteus M82860, X15768 108, 109 Oryza sativa X15901 265 Oscillatoria sp. X58359, X58360, X58361 (partial) 698, 699 Palmaria palmata Z18289 573 Pisum sativum M37430 602 X51598 79 M16874, M16862, M30826 (partial) 557, 617, 618 Porphyra purpurea L07257, L07258 516 Porphyridium sp. 34 Prochloron sp. X63141 555, 660, 665 Pylaiella littoralis M21373, X14873, X14874 404, 405 Pyrenomonas salina X55015 382 Sinapis alba M15915, X04182 502 Spinacia oleracea J01440, M21453 (partial) 56, 409 Spirodela oligorhiza X00014, X00015 (partial) 302 Synechococcus lividus X67091, X67092, X67093 (partial) 38 Zea mays M10720, Z00028 554 23S rRNA Alnus incana M75722 351 Anacystis nidulans X00512, X00343 (partial) 126, 334 Antihamnion sp. X54299 (partial) 384 Astasia longa X14386 569 Chlamydomonas eugametos Z17234 200, 657, 658 Chlamydomonas frankii X68905-X68909 658 Chlamydomonas gelatinosa Z15151 658 Chlamydomonas geitleri X68891, X68892 658 Chlamydomonas humicola X68921, X68922 658 Chlamydomonas indica X68893-X68898 658 Chlamydomonas iyengarii X68886, X68886 658 Chlamydomonas komma X68927-X68929 658 Chlamydomonas mexicana X68910-X68912 658 Chlamydomonas moewusii X68913-X68918 658 Chlamydomonas pallidostigmatica X68899-X68904 658 Chlamydomonas peterfii X68887, X68888 658 Chlamydomonas pitschmanii Z15152 658 Continued on following page 706 HARRIS ET AL. MICROBIOL. REV.

TABLE 1-Continued Taxon GenBank accession no(s). Reference(s) Chlamydomonas reinhardtii J01398, X01977, X16687, X16686 346, 521 Chlamydomonas starrii X68889, X68890 658 Chlamydomonas zebra X68919, X68920 658 Chlamydomonas sp. X68923-X68926 658 Chlorella ellipsoidea M36158; X05693, X03848 (partial) 726 Coleochaete orbicularis X52737 (partial) 394 Conopholis americana X59768 703 Cryptomonas (F X14504 (partial) 128 Cyanidium caldarium X54300 (partial) 384 Cyanophora paradoxa M19493 (partial) 287 Epifagus virginiana M81884, X62099 435, 712, 714 Euglena gracilis X13310, X12890 549, 730 Marchantia polymorpha M13809, X04465, X01647 312, 473 Nicotiana tabacum J01446, Z00044 560, 561, 627 Olisthodiscus luteus X15768 (partial) 108 Oryza sativa X15901 265 Palmaria palmata Z18289 573 Pisum sativum M37430 602 Pylaiella littoralis X61179, M21373 (partial) 405, 581 Spinacia oleracea M21453, X04977 (partial) 11, 409 Spirodela oligorhiza X00012, X00013 (partial) 304 Zea mays Z00028, X01365 148 4.5S rRNA Acorus calamus M36166 31 Allium tuberosum M35406 738 Alnus incana M75719 284 Apium graveolus M35404 738 Codium fragile M35276 176 Commelia communis M35407 738 Conopholis americana X58863 703 Dryopteris acuminata X01523 623 Gossypium hirsutum X63124 440 Hordeum vulgare M35405, M57605 80, 738 Jungermannia subulata M13808 691 Ligularia calthifolia M36165 31 Lycopersicon esculentum M33098 739 Marchantia polymorpha X04465, M13809 472, 473, 691 Marsilia quadrifolia X51641 421 Mnium rugicum M35056 652 Nicotiana tabacum J01446, V00161, J01891, J01451, X01277, Z00044 560, 561, 624, 625 Oryza sativa X15901 265 Osmunda regalis X51978 421 Pisum sativum M37430 602 Spinacia oleracea M10757, X04977 11, 332 Spirodela oligorhiza J01439 303 Triticum aestivum M10541 696 Zea mays M19943, Z00028, X01365 147, 148, 601 5S rRNA Alnus incana M75719 284 Anacystis nidulans X00343, X00757, M23834 94, 125 Astasia longa X14386 569 Chlamydomonas reinhardtii X03271 550 Chlorella ellipsoidea X04978 724 Conopholis americana X58863 703 Cyanophora paradoxa M32451, M33030 411, 412 Cycas revoluta X12787 743 Dryopteris acuminata X00758 629 Euglena gracilis bacillaris X00536 152 Euglena gracilis K02483, X12890 243, 296 Ginkgo biloba X51979 421 Glycine max X16736 23 Gossypium hirsutum X63124 440 Jungermannia subulata X00667 728 Juniperus media 663 Lemna minor X02714 144 Lupinus albus X65030 327 Marchantia polymorpha X00666, X04465 472, 473, 728 Continued on following page VOL. 58, 1994 CHLOROPLAST RIBOSOMES AND PROTEIN SYNTHESIS 707

TABLE 1-Continued

Taxon GenBank accession no(s). Reference(s) Nicotiana tabacum J01451, M10360, M15995, X01277, Z00044 144, 560, 561, 624-626 Oryza sativa X15901 265 Picea excelsa X63200 421 Pelargonium zonale X05551 146 Pisum sativum M37430 602 Porphyra purpurea L07259, L07260 516 Porphyra umbilicalis 664 Prochloron sp. K03159, X02637 380 Pylaiella littoralis X61179 580 Spinacia oleracea V00169, X05876 112, 491, 492 Spirodela oligorhiza J01439 303 Synechococcus lividus X02731 111, 113 663 Zea mays M19943, Z00028 147, 601

example, primary is generally over 70% 16S rRNA for chloroplast or cyanobacterial 16S rRNAs compared with that of E. coli and greater than 80% for chloroplast 16S rRNA The secondary-structure model of 16S rRNA based on compared with cyanobacterial 16S rRNAs. Gray (219) recog- comparative sequence analysis (231, 232, 236, 449, 463, 468) nized eight noncontiguous conserved primary sequences in 16S suggests a functional division into distinct 5', central, and 3' rRNA interspersed among nonconserved sequences. The pre- domains, corresponding in E. coli to residues 26 to 557, 564 to dicted secondary structures of these molecules are even more 912, and 926 to 1391, respectively, followed by a "3' minor conserved, and virtually all of the approximately 45 helices domain" from ca. 1401 to 1542 (Fig. 3; for a numbered E. coli postulated for the E. coli 16S rRNA (62, 462) are present in sequence diagram in similar format to the tobacco sequence chloroplast 16S rRNAs of Euglena gracilis, Chlamydomonas shown in Fig. 3, see references 231 and 235). Each of these species, tobacco, and maize (232, 512; also see below). Com- domains comprises helices and loops whose secondary struc- pensating base substitutions are often seen on the complemen- ture is phylogenetically conserved (219, 236). Models for the tary sides of predicted stem structures, strengthening the tertiary structure of the E. coli 30S subunit have been con- supposition that these structures are functional in vivo. Be- structed based on studies of RNA-RNA and RNA-protein cause of this high degree of structural conservation, rRNA cross-linking, immunoelectron microscopy, and neutron dif- genes have found extensive use in phylogenetic studies (78, fraction (58-61, 463, 465, 596). Functional analyses involving 219, 232, 235, 236, 342, 710). Comparative analyses of 16S (54, mutants, binding of tRNA and antibiotics, and assembly of 210) and 5S (144, 380, 663, 743) rRNA sequences support both ribosomal proteins with RNA in vitro indicate that codon- the probable origin of chloroplasts from endosymbiotic cya- anticodon recognition involves the 3' domain and terminal 3' nobacteria and the hypothesis that land plants derive from one minor domain. Three regions of the 16S molecule (E. coli nt branch of chlorophyte algae. Van de Peer et al. (665) have 518 to 533, 1394 to 1408, and 1492 to 1505) that show a compared 16S and 18S sequences from eukaryotic, archaebac- particularly high degree of primary sequence conservation terial, eubacterial, plastid, and mitochondrial ribosomes. Al- appear to have tertiary interactions related to decoding (468). though their analysis focused largely on mitochondrial origins, tRNA bound in the A site interacts specifically with the 3' their data also support the common ancestry of cyanobacteria domain and with residues in the "530 loop" (see reference 465 and . for review), whereas P-site-bound tRNA protects five sites in the central and 3' domains that are proposed to be clustered in General Characteristics of Chloroplast rRNA Gene Organization tRNA As in the eubacteria, chloroplast rRNA genes are normally 168 lie Al 23S 5s arranged in an operon transcribed in the order 16S-23S-5S _uI IE cyanobacteria, most algae (Fig. 2) (114, 320). In land plants, including some but not all tRNA ferns, approximately 95 nt homologous to the 3' terminus of 165 I 23S 4.5S 5S the E. molecule constitutes a rRNA coli 23S 4.5S molecule, land plants separated from the remainder of the 23S gene by a transcribed tRNA spacer, whereas in prokaryotes, all algae so far examined, 165 IleAla 7S 38 23S 5' 23S 3' 5S mosses, and the liverwort Marchantia the equiva- polymorpha, C. relnhardtll lent sequence is part of the 23S gene (47, 320). In C. reinhardtii, the sequences homologous to the 5' portion of the 23S gene of FIG. 2. Arrangement of the rRNA operons in land plants and bacteria and are divided into 7S and 3S algae, showing conservation of tRNAIle and tRNAMa within the spacer plants rRNAs, between the and 23S and variation in the that short that are removed from the 16S genes species separated by spacers precur- constitute the 23S molecule. In land the tRNA are rRNA plants, genes split sor rRNA posttranscriptionally (137). The large subunit by introns, whereas in all algae examined to date they are uninter- of C. eugametos comprises species (a and ,B) equivalent to the rupted. The region corresponding to the 3' end of the eubacterial 23S C. reinhardtii 7S and 3S rRNAs and two larger species (ry and 8) rRNA is a separate 4.5S rRNA in angiosperms, gymnosperms, and which together are equivalent to the remainder of the 23S some (but not all) ferns. Internal transcribed sequences and one or molecule (656). more introns interrupt the 23S genes of Chlamydomonas species (658). 708 HARRIS ET AL. MICROBIOL. REV.

central domain UA GA a^o a CMnGAA A c^^cAA a GUc 'aGGUGGCCUUUAAGGG-cCA CGA A U a-C cA-aU a0-C GO 0 spr - U.G u a G-C C-Q C - GA c-a U - AC ^ a GA G G a 0-C A A-U AaUUCUCCU~AA ACA AA cc U A AACCCUG0ACCCGGCGOUGGA CU A AAGC C 11I1II1I1 I .II I .II II IIIIIlIi CAC CUGCCGCCU GA UUUUUC GU/.UAG 3' domain UCGOGACC A 0. A0! CU UAAA G~~~~~A A

helix 17

5' domain

isr

0

5, nr

A GUC ACOGGAAGUG I - I I In I I I I- a COO U0ACCUUUoU a helix 6 G a C 3' minor domain

tobacco 16S rRNA

FIG. 3. Secondary structure of tobacco 16S rRNA, showing the major functional domains and sites of antibiotic resistance (Table 2; sr, streptomycin; spr, spectinomycin; nr, neamine/kanamycin). Sequence from the Ribosomal Database Project, courtesy of Robin Gutell. the tertiary structure. Many of the same sites, which are all in chloroplast 16S rRNAs deviate from the E. coli model are in highly conserved regions of the 16S molecule, also interact with the 5' domain between nt 198 and 220 (numbering according antibiotics that block protein synthesis at the level of the 30S to the E. coli sequence [462]), where chloroplast rRNAs have subunit (424, 467, 537; also see below). a shorter helix 10 than E. coli does, and between 455 and 477, The principal regions in which the secondary structures of where E. coli has a well-defined helix (the upper part of helix VOL. 58, 1994 CHLOROPLAST RIBOSOMES AND PROTEIN SYNTHESIS 709

17 of Brimacombe et al. [59]) that is lacking in cyanobacteria teria and plastid rRNAs, as does domain V. The break and chloroplasts. Helix 6 is also shorter in chloroplast and between the 23S rRNA and 4.5S rRNA of land plants occurs in cyanobacterial 16S rRNAs than inE. coli. Raue et al. (512) domain VI. noted eight regions where secondary structure is conserved but 5S rRNA primary sequence is highly variable. These eight regions are also sites of variation in secondary structure when the 16S Their short length and relatively high degree of evolutionary rRNAs of chloroplasts and eubacteria on the one hand are conservation have madeSS rRNA molecules frequent subjects compared with small-subunit rRNAs of mitochondria and for phylogenetic studies (see e.g., references 117, 270, 580, 663, eukaryotic cytoplasmic ribosomes on the other. 664). They have also proved useful for computer modeling of secondary and tertiary structure, including chemical reactivity 23S rRNA and accessibility of bases, and possible protein binding (67, 524, 525, 693). A numbering scheme applicable to both pro- The secondary-structure model for the E. coli 23S rRNA karyotic and eukaryoticSS rRNAs, proposed by Erdmann and published by Gutell and Fox (233) consists of six domains Wolters (157), defines five loops (a to e) and five helices (A to comprising a total of 95 helices (Fig. 4; for a numberedE. coli E). In a compilation of sequences in the Berlin RNA Data- sequence diagram in similar format to the tobacco sequence bank, Specht et al. (583) included representations of the shown in Fig. 4, see references 234 and 235). Domain V is the common secondary structure of eukaryotic and prokaryotic SS principal site of tRNA binding to the50S subunit (60, 426). rRNAs, which are differentiated into five structural groups The central loop of this domain is involved in the peptidyl- primarily on the basis of variability in one (D) of the five transferase center and is the site of mutations conferring helices. PlastidSS rRNAs are grouped in this classification with resistance to erythromycin, lincomycin, and chloramphenicol those of eubacteria and land plant mitochondria (mitochon- (see below). Some tRNA interactions are found in domainsII dria of other taxa lackSS rRNA). Plastid and cyanobacterial SS and IV. When bound to the P site, tRNA also interacts with the rRNAs are distinguished from those of most other eubacteria 3' terminus of the 23S molecule (426). EF-G binds specifically and mitochondria by a single-base insertion in helix C and a to position 1067 in the 23S molecule, a region identified with GTP deleted base in loop c (157). Of the 121 nt of the typical SS hydrolysis (465). EF-Tu protects residues in the 2660 loop. rRNA, 110 are identical in nearly all angiosperms and gymno- Rau6 et al. (512) identified 18 variable regions in 23S RNAs sperms, 73 are conserved in ferns and liverworts as well, and 29 based on comparisons of eubacterial, organelle, archaebacte- are identical in all plastids so far sequenced with a few singular rial, and eukaryotic large-subunit rRNAs, including the cya- exceptions. The colorless flagellate Astasia longa and the red nobacterium Anacystis nidulans and chloroplast 23S rRNA alga P. umbilicalis are somewhat divergent compared with from Chlorella ellipsoidea, Marchantia polymorpha, tobacco and Euglena gracilis and P. purpurea, respectively; 4 nt are altered maize. Of these 18 variable regions,5 are significantly different in one or both of the two parasitic plants Conopholis americana in chloroplasts compared withE. coli, while in the remaining and Epifagus virginiana compared with all other angiosperms; 13 regions, chloroplast rRNAs resemble those of eubacteria and the sequence submitted to GenBank for cotton, Gos- but may differ from those of archaebacteria, mitochondria, and sypium hirsutum (440), is missing 2 nt but is otherwise identical eukaryotic cytoplasmic ribosomes. Somerville et al. (581) have to that of tobacco in all but two residues. Vogel et al. (671) published a secondary-structure map of the 23S rRNA from reported that SS rRNA from spinach chloroplasts could be the brown alga Pylaiella littoralis which resembles the cya- incorporated into biologically active 50S ribosomal subunits nobacterial (Anacystis) molecule much more closely than it assembled in vitro from Bacillus stearothermophilus proteins resembles those of land plants or green algae. Cladistic analysis and 23S rRNA. of the 23S rRNA sequence produced a tree in which cyanobac- terial and plastid sequences were clearly delineated from all Introns in rRNA Genes other eubacterial sequences and in which the chromophyte algae (as represented by Pylaiella littoralis) and Euglena gracilis A survey of 23S rRNA genes from 17 Chlamydomonas formed a common branch. species representing most of the taxonomic groups defined on In domain I, cyanobacterial and chloroplast 23S rRNAs lack morphological and biochemical grounds (159, 538) revealed a helix 8 of E. coli (nt 131 to 148, variable region V1) and have total of 39 group I introns inserted at 12 different positions, an insertion between helices 13 and 14 (E. coli nt 271 to 365, some of which were unique to Chlamydomonas species (656- variable region V2) which can be folded into a helix (512). In 658). However, no correlation was found between intron domain II, variable regions V4 and V7 (nt 636 to 655 and 1020 distribution and a phylogeny for these 17 species based on to 1029, respectively) are highly conserved among eubacteria primary sequence of their 23S genes. Most of the intron and chloroplasts, while 3 nt (nt 931 to 933) in E. coli V6 are insertion sites identified in this study are in highly conserved replaced by a loop of5 to 20 nt in chloroplasts. Region V8 (nt regions of the genome, which tend to be exposed in the 1164 to 1185) is conserved in E. coli, Anacystis nidulans, and assembled ribosome. This is also true of the single intron in the most chloroplast 23S rRNAs but is the site of a possible 243-nt 16S gene of C. moewusii, which lies within the 530 loop, a part intron in Chlorella ellipsoidea (726). Gutell and Fox (233) have of the translational fidelity domain. In contrast, internal tran- suggested that this insertion may actually be a part of the scribed spacers, which have also been identified in rRNA genes rRNA rather than the only known instance of an intron of bacteria and organelles, occur within regions of variable inserted in a variable rRNA region. primary sequence and secondary structure (224). When these Domain III comprises variable regions V9 to V12, of which sequences are processed out of the pre-rRNA molecule, the Vii (E. coli nt 1521 to 1542) is the most diverse in chloroplast mature sequence is not religated, resulting in a fragmented 23S rRNAs. Some (but not all) chloroplast rRNAs have lost rRNA. Three internal transcribed spacers, found at equivalent part of helix 54, and helix 55 in Chlorella ellipsoidea and Z. positions in the Chlamydomonas taxa studied by Turmel et al. mays contains insertions compared with E. coli; however, in (656-658), result in fragmentation of the 23S rRNA into four other chloroplast genomes, this helix is similar in size to that of mature rRNA species, ao, ,, -y, and &. E. coli. Domain IV shows strong conservation among eubac- The single group I intron in the 23S gene of C. reinhardtii 710 HARRIS ET AL. MICROBIOL. REV.

Ua AAAAU c B c A u VI BAA I U-A G tcc A AUUUC a eUAAGAAG A I - *I I II I I I II I A AcUoUUUC%cccuu OGAAUBCAAA u c c UUA Ue c GTPase a UCOB AGGCGCCu G ,,|"III UA ABCC a 0UC0CB center OAA A _UBAAG U-A Uu-CA AA - c AU u~~~~~~~~~A~~~~~~~~ aAU u a u u 0-C 0-C 6-C O-C A A A-U U00 V9 AA -1B ViOA BA% 0AUCA GOCCBUC 0ACQ%%.IA aa4AusUCG ZqGCCCCCUUBUUBa CC0ABzG °scU AAABBCPUAI1111IIIIII,I*IUBUC BOAI r BuuCUCI AcC Aa CCBA CUAAAU UG.LCACAG A ACC A V7 GUCAUA AAAf

C II 111111- A UUBG OCCUCCU' 'ac GUUUAA AACu1G1 AB-U A[q ~~~domainV12 III A C AAC U C-BUB' CCAA .UE

_sC-B c f U A I UI-C_ _CauuC-Gc bAA3'fn

V6 domain I

V3

domain 11

AA CAA a c u AA A e C=U U-A a-B B-C AAC-BGBUUBUC-B V2 -QU tobacco 23S rRNA, 5' a A oil 11|1 11, AAA' 0-2

._

FIG. 4. Secondary structure of tobacco 23S rRNA, showing major functional domains and sites of antibiotic resistance (Table 2). Variable regions are numbered according to the system of Raue et al. (512). Sequence from the Ribosomal Database Project, courtesy of Robin Gutell. VOL. 58, 1994 CHLOROPLAST RIBOSOMES AND PROTEIN SYNTHESIS 711

U C C-aU * G C-C UC- G-C a-CA AG A -C AICUAC uu V5 A G-CA domain IV LL 3UGG domainIV C,1C UCACCTf CU U- UAGAG c-a U-A U-A c-aC-a a-C GGAG G u GA GA A A u.G 0Uu U-A a V13 tu V16 a G-CUA Accuu U UCUCGGAC G-CC U -III I * I°I C G U GC A -UGACAG U-A Gu G0-C- c o A CCAG aa UC-aU aC A A C GOA AG U-A A A CG c-a c c U-A C - a G C-O U U I-C U-A Uuca AA- U -c U: c ACUGC. C C I I I III AUaUUC a G *U GU C-Gc a 1 G- ACG G U A-AA U : A-: C aC U-A0U-AG CG-CG-CUA U-A- AGUCCA-U aUu U-AA A A a AUA C A A AAC-G G_.CGUGaO CC

CAC-GGAU AU AA C A GGCUGAUCUUCCCCACCU peptidyl 1I.1i1i1iI It C transferase caUC GAAC A a Ac domain VI UU u C u

3A aG c a a U.a Ac 5' 4.5S UC C=a A CA- U cA UC CAUGG A G

_A G-C ACGGCGAG Ua-C A 11111 A C-a U%GCCGA Gc AAGCCACCUGUGGCUG U A UUAU BC-UA 'A A-UA C UtU U U-A A G0GACCUUGUA CC--G CAUU C-G A -U C=G C-G ~~~U a-*U C .A C' CU Aa-c u 'C UUG GUaA Ac tobacco 23S rRNIA, 3 0C -GUa Aa

C0 A U GA A-U U aG C-C U-A CGU

(522) is mobile (142) and encodes a double-stranded splicing in vitro (637). The ac-20 nuclear gene mutant of C. (I-CreI) which has been purified and shown to have a 19- to reinhardtii, which was initially characterized as deficient in 24-bp recognition sequence in this gene (143, 638). The chloroplast ribosomes (see reference 247 and references there- enzyme makes a 4-bp staggered cut just downstream of the in), has been found to accumulate unspliced precursor rRNA intron insertion site and will tolerate single- and even multiple- molecules, as well as unspliced precursors of the chloroplast- base-pair changes (143). This intron can undergo autocatalytic encoded psbA gene (258, 259). 712 HARRIS ET AL. MICROBIOL. REV.

The 23S rRNA gene of C. eugametos, a species now thought disrupted by an inversion of a 5-kb region with a breakpoint to be only distantly related to C. reinhardtii, contains six group between the two tRNAs, so that the 5S, 23S, and tRNAMa I introns and three internal transcribed spacers (657). One genes constitute a second operon on the opposite strand from optional 955-bp group I intron in the 23S gene of C. eugametos the 16S and tRNAIle genes (723, 725). also appears to be mobile and is transmitted to all progeny of crosses with the interfertile species C. moewusii, which lacks this intron (347). The 402-bp group I intron in the 16S rRNA tRNAs Flanking the rRNA Operons gene of C. moewusii likewise can be transmitted in crosses to isolates of the sibling species C. eugametos that lack this intron Genes encoding tRNAs are also often found in regions (140). Transmission of these introns is often accompanied by flanking the rRNA operons, but their presence and identity are coconversion of flanking DNA polymorphisms. The mobile much more variable than for the two tRNA genes in the intron in the 23S encodes a double-stranded DNA 16S-23S spacer. There is a tRNAVa" proximal to the 5' end of gene the 16S rRNA gene in all land plants examined (114). This endonuclease activity (I-CeuI) which has a 19-bp recognition which the for the rRNA is site centered around the insertion site. I-CeuI produces a gene, precedes promoter operon, staggered cut 5 bp down from the insertion site (200, 406, 407). not present in C. reinhardtii or C. moewusii, nor is it found of of the E. coli rm or in of the The 23S rRNA gene of C. humicola has a group I intron, upstream any operons any sequences to date. In the ChLSU-1, inserted at a site in the peptidyltransferase loop and cyanobacterial Euglena gracilis, a 218-amino-acid endonuclease In- equivalent tRNAVal is in a gene cluster distant from the rRNA encoding putative (96). and a is found 5' to the 16S trons have been found at this site only in a few Chlamydomonas operons (477), pseudo-tRNAIle species (658). gene (479). Turmel et al. (658) discuss the alternative possibilities for In land plants the 5S rRNA gene is typically followed by a transfer of I introns from one site to another within a tRNAN9 gene in the same orientation and by a tRNA,sn gene group on the opposite strand (118, 297, 298, 305, 557). In maize, genome. Intron-encoded endonucleases could effect such a extension have shown that the transfer at the DNA level (139); alternatively, a reversal of primer experiments tRNA'9 followed reverse of the recom- gene, which is separated from the 5S gene by a 252-bp spacer, self-splicing by transcription is cotranscribed with the rRNA operon (118). This operon and bined RNA could occur, followed by integration into DNA by In . The latter mechanism requires the tRNA gene, which is distal to tRNAArg by 253 bp on the are to share a common only a short target site that can pair with the 5' intron sequence opposite strand, thought terminator called the internal guide sequence (718) and would be consis- region consisting of a palindromic sequence which can be tent with the position of intron insertion sites in exposed rRNA folded into hairpin structures on both strands. regions in the ribosome in the Chlamydomonas species exam- ined by Turmel et al. (658). Antibiotic Resistance Mutations in the Chloroplast rRNA Genes The 16S-23S Spacer Many antibiotics that inhibit bacterial protein synthesis bind The spacer regions between the 16S and 23S rRNA genes in specifically to the 16S or 23S rRNA molecules (102, 424, 425), chloroZlasts and cyanobacteria contain tRNAIleGAU and and mutants resistant to these antibiotics have been shown to tRNA aUGcmas do the E. coli rmA, mD, and rmH operons result from single-base-pair changes in evolutionarily con- (301, 436, 697, 735). In E. coli and cyanobacteria, the 16S-23S served regions of the genes encoding these RNAs in bacteria, spacer is short (<550 bp), but in land plants and charophyte mitochondria, and chloroplasts (Table 2). Streptomycin resis- algae it is 1 to 2 kb or more, largely because of the presence of tance can result from changes at several nucleotides clustered type II introns in the two spacer tRNA genes (110, 311, 320, in three sites in the 16S chloroplast rRNA molecule of land 394, 628). In all other algae so far examined, these spacer plants and green algae (equivalent to E. coli residues 13, 523 to tRNAs are uninterrupted (108, 216, 384, 405, 551, 725). In C. 525, and 912 to 915). Although these three sites are widely reinhardtii, an 1,100-bp region between the 3' end of the 16S separated in the primary sequence, they interact with the same gene and tRNAIle contains short dispersed repeat elements in subset of ribosomal proteins and are thought to be in close direct and inverted orientations, which are capable of pairing proximity in the assembled 30S subunit of E. coli (603). to generate extensive secondary structure in the precursor Spectinomycin resistance has been shown to result from mu- RNA (551). Similar repeat elements are found elsewhere in tations at the bases of the chloroplast 16S rRNA equivalent to intergenic regions of the chloroplast genomes of C. reinhardtii E. coli residues 1191 to 1193 and at the base equivalent to and the interfertile species C. smithii, and variations in their residue 1064, which pairs with 1192. Neamine and kanamycin numbers are responsible for most of the restriction fragment resistance in C. reinhardtii can result from mutations at the length polymorphisms between the chloroplast genomes of chloroplast 16S rRNA nucleotides equivalent to E. coli resi- these isolates (50, 250, 486). The absence of these repeats in dues 1408 and 1409. In E. coli, binding of aminoglycoside chloroplast rRNA operons of other , including C. antibiotics to this region has been demonstrated (424, 717), eugametos and C. moewusii, and their variation in number in and site-directed mutagenesis of these and neighboring bases the 16S-23S spacer region between C. reinhardtii and C. smithii has been used to obtain a number of mutants (116). Because suggest they are not essential for processing of the rRNA the E. coli genome has seven rm operons, antibiotic resistance precursor. mutations must be selected by expression of cloned rRNA Although the 16S and 23S rRNA genes in the plastid operons on high-copy-number (570, 603). In contrast, genome of the colorless euglenoid flagellate Astasia longa are an efficient copy correction mechanism involving the inverted highly homologous to those of Euglena gracilis, the spacer repeat ensures that newly occurring 16S mutations can spread between these genes appears to lack tRNAIle and tRNAAla to both rRNA cistrons in the chloroplast genome (50). (569). The chloroplast genome of Chlorella ellipsoidea was also Erythromycin resistance mutations in the large subunit reported to lack the spacer tRNAAla (724), but subsequent rRNA are known in bacteria, in mitochondria of Saccharomy- analysis has shown that the rRNA operon in this alga has been ces cerevisiae and mammalian cells, and chloroplasts at the VOL. 58, 1994 CHLOROPLAST RIBOSOMES AND PROTEIN SYNTHESIS 713

TABLE 2. Antibiotic resistance mutations in chloroplast rRNA and ribosomal protein genes compared with analogous mutations in E. coli and mitochondria Taxon' Nucleotides Reference(s) 16S rRNA mutations to streptomycin resistance E. coli wild type 4 JUGAAGAGUUUGAUCAUG 21 66 E. coli mutant 4 . A . 21 493 E. coli mutant 4 . C . 21 493 C. reinhardtii wild type 5 AUGGAGAGUUUGAUCCUG 22 137 C. reinhardtii mutant 5 . G . 22 251 E. coli wild type 517 GCCAGCAGCCGCGGUAAU 534 66 E. coli mutant 517 ...... C...... 534 420 Nicotiana plumbaginifolia wild type 465 GCCAGCAGCCGCGGUAAU 482 729 N. plumbaginifolia mutant 465. U. 482 643, 644 Nicotiana tabacum wild type 464 GCCAGCAGCCGCGGUAAU 481 646

N. tabacum mutant 464 ...... U ...... 481 184 C. reinhardtii wild type 468 GCCAGCAGCCGCGGUAAU 485 137

C. reinhardtii mutant 468 ...... C...... 485 251 C. eugametos wild type GCCAGCAGCCGCGGUAAU 199

C. eugametos mutant ...... C 199 E. coli wild type 905 UAAAACUCAAAUGA 918 66 E. coli mutant 905 . . 918 431 E. coli mutant 905. G. 918 343 E. coli mutant 905 . U 918 493 E. coli mutant 905 . C 918 37 E. coli mutant 905 . ... 918 493 E. coli mutant 905 . ...G 918 343 Euglena gracilis wild type 869 UGAAACUCAAAGGA 882 217 E. gracilis mutant 869. U. 882 428 Mycobacterium tuberculosis 858 UAAAACUCAAAGGA 871 131 M. tuberculosis resistant isolate 858. G. 871 131 Nicotiana plumbaginifolia wild type 854 UGAAACUCAAAGGA 867 729 N. plumbaginifolia mutant 854. U . 867 643, 644 N. tabacum wild type 853 UGAAACUCAAAGGA 866 646 N. tabacum mutant 853. A. 866 160 N. tabacum mutant 853. U. 866 184 C. reinhardtii wild type 849 UGAAACUCAAAGGA 862 137 C. reinhardtii mutant 849. U. 862 251 C. reinhardtii mutant 849 . C 862 251 C. reinhardtii mutant 849 .G 862 251 16S rRNA mutations to spectinomycin resistance N. tabacum wild type 1006 GCUGUCGUCAGC 1017 646

N. tabacum mutant 1006 ...... 1017 182 N. tabacum wild type 1133 GGAUGACGUCAAGU 1146 646

N. tabacum mutant 1133 ...... 1146 620 N. tabacum mutant 1133 ...... U.1146 620

N. tabacum mutant 1133 ...... 1146 182 C. reinhardtii wild type 1118 GGAUGACGUCAAGU 1131 137 C. reinhardtii mutant 1118 ...... 1131 249, 251 C reinhardtii mutant 1118 ...... 1131 249, 251 C. reinhardtii mutant 1118 ...... 1131 249, 251 E. coli wild type 1186 GGAUGACGUCAAGU 1199 64 E. coli mutant 1186 ...... U.1199 26, 387, 570 E. coli mutant 1186 ...... G .1199 26, 387 E. coli mutant 1186 ...... A.1199 26, 387 Zea mays (naturally resistant) 1132 GGAUGAGGCCAAGU 1145 554 N. tabacum wild type 1326 GUUCCCGGGCCUUGUAC 1341 646 N. tabacum mutant 1326 ...... 1341 620 16S rRNA mutations to neamine and kanamycin resistance C. reinhardtii wild type 1332 CGCCCGUCACACCAUGGA 1349 137 C. reinhardtii mutant 1332 ...... G.1349 251

C. reinhardtii mutant 1332 ...... 1349 251 23S rRNA mutations to erythromycin and/or lincomycin resistance C. reinhardtii wild type 2007 CUGGACAGAAAGACCC 2022 346

C reinhardtii mutant 2007 ...... 2022 251

C. reinhardtii mutant 2007 ...... 2022 251 E. coli wild type 2050 CAAGACGGAAAGACCC 2065 65

E. coli mutant 2050 ...... 2065 158 Continued on following page 714 HARRIS ET AL. MICROBIOL. REV.

TABLE 2-Continued Taxona Nucleotides Reference(s) 2

E. coli mutant 2050 ...... 2065 570 E. coli mutant 2050 ...... 2065 132 Nicotiana plumbaginifolia wild type CUGGACAGAAAGACCC 99 N. plumbaginifolia mutant ...... G...... 99 N. plumbaginifolia mutant ...... 99 Saccharomyces cerevisiae mitochondria wild type 1943 GCAGACGGAAAGACCC 1958 See 100 S. cerevisiae mutant 1943 . . G...... 1958 See 100 E. coli wild type 2601 CAGUUCGGUCCCUAUC 2616 65 E. coli mutant 2601 ...... u ..... 2616 667 C. reinhardtii wild type 2559 CAGUUUGGUCCAUAUC 2574 346 C. reinhardtii mutant 2559 ...... U ..... 2574 251 C. reinhardtii mutant 2559 ...... G..... 2574 251 C. moewusii wild type CAGUUUGGUCCAUAUC 199 C. moewusii wild type ...... 199 S. cerevisiae mitochondria wild type 2775 CAGUAUGGUUCCUAUC 2790 See 100 S. cerevisiae mutant 2775 ...... G 2790 See 100 S. cerevisiae mutant 2775 ...... 2790 100 23S rRNA mutations to chloramphenicol resistance E. coli wild type 2499 CUCGAUGUCGG 2509 65 E. coli mutant 2499 ...... 2509 158, 425 C. reinhardtii wild type 2509 CUCGAUGUCGG 2519 346 C. reinhardtii mutant 2509 ...... 2519 208 S. cerevisiae mitochondria wild type 2672 CUCGAUGUCGA 2682 See 158 S. cerevisiae mutant 2672 ...... 2682See 158 S. cerevisiae mutant 2672 ...... 2682 See 158 S12 mutations to streptomycin resistance and dependence E. coli wild type 38 TTTPKKPNSA 47 707 E. coli mutant (sr) 38 .... N. 47 188 E. coli mutant (sr) 38 . Q..Q. 47 190 E. coli mutant (sr) 38 .... R. 47 188 E. coli mutant (sr) 38 .... T. 47 190 C. reinhardtii wild type 38 TVTPKKPNSA 47 364 C. reinhardtii mutant (sr) 38 .... T. 47 364 E. coli wild type 83 GGRVKDLPGV 92 707 E. coli mutant (sd) 83 .S.. 92 285 E. coli mutant (sr) 83 .... R. 92 188 E. coli mutant (sd) 83 . L 92 662 E. coli mutant (sd) 83. D. 92 285 C. reinhardtii wild type 83 GGRVKDLPGV 92 364 C. reinhardtii mutant (sd) 83 . L 92 364 N. plumbaginifolia wild type 83 GGRVKDLPGV 92 276 N. plumbaginifolia mutant (sr) 83 .... R. 92 276 N. tabacum wild tpe 83 GGRVKDLPGV 92 561 N. tabacum mutant (sr) 83 .S 92 191 a sr, streptomycin resistance; sd, streptomycin dependence.

positions equivalent to E. coli nt 2057 to 2058 (the yeast rib3 RIBOSOMAL PROTEINS locus) and 2611 (yeast rib2) (Table 2). Some of the erythro- mycin-resistant mutants of C. reinhardtii are cross-resistant to Number and Nomenclature lincomycin. A lincomycin-resistant mutant of Nicotiana plum- Recent reviews provide an overview of chloroplast ribo- baginifolia has also been identified at the base equivalent to E. somal proteins and the genes that encode them (36, 377, 379, coli nt 2032. Chloramphenicol resistance in C. reinhardtii 606, 608, 609, 615). For a concise summary of structure and results from a nucleotide substitution at a position equivalent function of individual ribosomal proteins, with emphasis on the to E. coli nt 2504 (208). Chloramphenicol resistance mutations E. coli ribosome, see the review by Liljas (358). Lindahl and at this site are also known in mitochondria of yeast (the rbl Zengel (360) provide a review of bacterial genes for ribosomal locus) and mammals. All three regions of conserved sequence proteins. Chloroplast ribosomal proteins were initially identi- together form a loop known to be involved in peptidyltrans- fied from various plants and algae by one-dimensional and ferase activity in E. coli (464). two-dimensional gel electrophoresis and were numbered ac- Chloroplast antibiotic resistance mutations have been used cording to their migration on these gels, a function of charge as markers in generation of transgenic tobacco plants by using and/or molecular mass, depending on the gel system. Natural somatic cell fusions (427) and in chloroplast transformation of variations in the physical properties of ribosomal proteins Chlamydomonas species (48, 49, 453) and of tobacco (390, 619) themselves, together with differing electrophoretic conditions by using biolistic techniques. used for their separation, have meant that no two numbering VOL. 58, 1994 CHLOROPLAST RIBOSOMES AND PROTEIN SYNTHESIS 715 schemes are the same. Even the well-characterized ribosomal seen in the Marchantia mitochondrial genome (630). The rps4 proteins of E. coli are not numbered according to their and rpsl4 genes are also found in chloroplast genomes of land migration on two-dimensional sodium dodecyl sulfate-poly- plants but are relocated outside the ribosomal protein cluster. acrylamide gels. Since most of the chloroplast ribosomal In some legumes, rp122 has been removed from this cluster and proteins identified on gels have not yet been correlated with relocated to the nucleus (193), and in a number of dicots the sequenced genes, -specific terminologies are some- chloroplast rp123 gene is disrupted and probably nonfunctional times used, e.g., the numbering system of Mache et al. (378) (see below). The rp122 gene is also missing from this operon in and Dorne et al. (119, 120) for spinach, the system of Capel Chlamydomonas species (43, 277). In Euglena gracilis (242) and and Bourque (73) for tobacco, and that of Schmidt et al. (547) in both C. reinhardtii and C. moewusii (43, 277), the ribosomal for C. reinhardtii. When used here, such designations will be protein gene cluster also contains rplS, which does not appear given in quotation marks as "L-13," etc. However, in cases in to be present in the chloroplast genomes of land plants. which equivalence has been established, designations of the However, the Euglena operon lacks rpsll, which is now in a chloroplast ribosomal proteins and the genes encoding these separate operon with rps4 (242, 597). The Chlamydomonas proteins have been changed to indicate the E. coli ribosomal operons lack rps3, but open reading frames with homology to proteins to which they correspond. For example, seven of the rps3 are found elsewhere in the chloroplast genome (see ribosomal proteins from chloroplast ribosomes of spinach have below). In C. moewusii, the large ribosomal protein cluster has been purified by Schmidt et al. (540) and shown by N-terminal been disrupted by a rearrangement such that rp123, rpl2, and sequencing to be equivalent to seven E. coli proteins (S12, S16, rpsl9 are separated from rplJ6, rpll4, rplS, and rps8 by about 42 S19, L20, L32, L33, and L36), whose homologs are encoded by kb (43). The genes encoding S17, L24, and L15, which are part chloroplast DNA. Those chloroplast and nuclear genes encod- of these operons in bacteria, have been identified in the ing chloroplast ribosomal proteins corresponding to those ofE. nuclear genomes of certain land plants (153, 196, 640). The coli are designated rps- or rpl- followed by numbers equivalent remaining genes of these E. coli ribosomal protein operons to the similar bacterial ribosomal protein designation (S1 to (encoding proteins L17 and L30) have not been identified with S21 for small-subunit ribosomal proteins and Li to L36 for plastid equivalents thus far. large-subunit ribosomal proteins [241, 606]). Thus, rps4 en- In E. coli, the genes encoding S12, S7, and the elongation codes protein S4 and corresponds to E. coli rpsD, rpl2 encodes factors EF-G and EF-Tu constitute a fourth operon in the str protein L2 (E. coli rplB), etc. cluster (Fig. 5). This operon has undergone several alterations Previous estimates of the number of chloroplast ribosomal in the course of plant evolution. It persists intact in cyanobac- proteins in the small and large subunits have been in the range teria (422, 641), but the fiusA gene encoding EF-G is absent of 22 to 31 and 32 to 36, respectively (73, 119, 156, 495, 547), from all chloroplast genomes analyzed so far and has presum- i.e., at least as many as in E. coli, in which 21 and 33 proteins ably been relocated to the nucleus. In Cyanophora paradoxa, have been identified in the small and large subunits, respec- the cyanelle str operon includes rpsl2, rps7, tufA, and rpslO, tively. Part of the variability in these estimates is undoubtedly which are processed from a primary transcript into two dicis- the result of differing isolation and electrophoretic conditions. tronic mRNAs (68, 69, 368). The rpslO gene is also down- In some circumstances these factors may cause certain pro- stream from tufA in Porphyra and Cryptomonas species (517). teins, particularly those of higher molecular weight, to be In the Euglena chloroplast, the rpsl2 and rps7 genes constitute excluded from gels (see references 510 and 547 for discussion). one operon and the tufA gene remains adjacent but is sepa- rately transcribed (430). In land plants, where tufA is a of Chloroplast Ribosomal Protein Genes gene (15), the rpsl2 gene has been split, with the second and third exons remaining proximal to rps7 and the first The first suggestion that chloroplast genomes might encode encoded separately downstream from rpl20 (183, 187, 649). chloroplast ribosomal proteins came from labeling experi- Lew and Manhart (352) have recently reported that the rpsl2 ments carried out in the presence of inhibitors specific for gene is also split in a green alga, Spirogyra maxima. This alga is either chloroplast or cytoplasmic protein synthesis. These believed to represent a relatively early stage in the charophyte studies led to the then remarkable conclusion that in land lineage leading to land plants. The rpsl2 mRNA is assembled plants, Euglena gracdiis, and C. reinhardtii about one-third of the by trans-splicing (264, 313, 737). In three species of the chloroplast ribosomal proteins were themselves synthesized- on angiosperm genus Anemone, the second rpsl2 intron has been chloroplast ribosomes, with the remainder being made on cyto- lost secondarily, in conjunction with expansion of the inverted plasmic ribosomes and imported (121, 156, 181, 239, 495, 547). repeat and several inversions within the chloroplast genome With a few notable exceptions, the same subset of ribosomal (269). proteins is encoded in the chloroplast genome of each land In two Chlamydomonas species, the rpsl2 operon has been plant examined, while marked variations occur in certain algal further disrupted. In C. reinhardtii, the uninterrupted rpsl2 groups (see below). The genes encoding many of these pro- gene (364) is separated from tufA by about 40 kb and is teins are arranged in clusters that are clearly remnants of the cotranscribed with the psbJ and atpI genes encoding photosyn- ribosomal protein operons of eubacteria (Fig. 5) (see refer- thetic proteins (253, 572). The entire rps7 gene is located in the ences 36, 377, 606, and 608 for additional discussion). The other single-copy region about 50 kb away, 5' to and cotrans- Porphyra plastid genome has the most complete version of cribed with the atpE gene (253). In C. moewusii, rpsl2, rps7, these operons found to date (Fig. 5; Table 3) (514, 517). Most and tufA are also widely separated; both rps7 and rpsl2 have of the same genes are also present in the cyanelle genome, but been completely sequenced and are uninterrupted (655). the operon has been broken into three pieces (598). In land Most of the remaining chloroplast-encoded genes for ribo- plant chloroplasts the largest cluster contains the genes for somal proteins are transcribed either separately or in operons ribosomal proteins L23, L2, S19, L22, S3, L16, L14, S8, L36, that also contain genes for photosynthetic proteins (606, 608). and S11 and the RNA polymerase gene rpoA in the same order The rp133 and rpsl8 genes are cotranscribed in land plants, that they appear in the E. coli S10, spc and a operons, which whereas the corresponding genes in E. coli are each part of a are part of the str cluster (Fig. 5; see Table 3 for references). A different operon. The rpl21 gene, which is monocistronic in the similar organization of genes encoding ribosomal proteins is Marchantia chloroplast genome (312), has not been found in 716 HARRIS ET AL. MICROBIOL. REV.

str S10 E. coil operons 812 LI fE tf 810 18D 123 810L225183 IL16 129

Cyanophora L 810L3 L2 81L22 83 I L16 8 812DEJuf A Porphyra 81 IEI 81018L4L23 L2 EjJiJ1 83 16 12 A Euglena 812j[E E.J C. relnhardtll i] Marchantla Ri~JEE] 819 1 83 1L16 Epifagus 812' 123 12 [ [GITiSi3| 16 1! 123T~J 19 i~jr~I3I j1 Nicotlana 8 i

apC alpha E coil operons 1~~~~ 15ER|814 88 16118 85 IE130 &K1S lIlI 136 I 8131111 || S4 IiFPOAII 117 I Cyanophora EjE7 rEli 88 16 116 85 ElbElJ_rpo Porphyra 114 ELE1688 188 EREEJ~~ rpo

Euglons E1 E _JE

C. relnhardtll EE1 Marchantla Epifagus _136 | Nicotlana ES FIG. 5. Conservation of ribosomal protein gene clusters in chloroplast genomes, showing retention of some (but not all) genes of the closely adjacent str, S10, spc, and a operons of E. coli (12), in Cyanophora paradoxa (598), P. purpurea (514, 517), Euglena gracilis (243), C reinhardtii (43, 277), Marchantia polymorpha (471), Epifagus virginiana (712, 714), and Nicotiana tabacum (560). Shaded boxes indicate genes that have been lost from the corresponding operon but have been identified elsewhere in a given plastid genome. For example, rps7 and tuf4 are present in the C reinhardtii chloroplast genome but have become separated from rpsl2. In Cyanophora paradoxa and P. purpurea, the operon begins with the rpl3 gene (A) and ends with the rpsl2, rps7, tufA, and rpslO genes (517, 598). The rpsl2 gene in land plants is split, and the 3' portion of the gene remains proximal to rps7. the Euglena, rice, or tobacco chloroplast genomes (243, 265, distinct genes for chloroplast ribosomal proteins that show no 560) and has been identified as a nuclear gene in spinach (408, obvious sequence similarity with any bacterial ribosomal pro- 578). Conversely, rpsl6 is a chloroplast gene in all angiosperms tein have been found in the nuclear genomes of pea and so far examined and in Euglena, Cyanophora, and Porphyra spinach (192, 290, 741). species (Table 3) but is absent from the Marchantia and Pinus In general, the chloroplast-encoded ribosomal proteins show thunbergii chloroplast genomes (614, 654). greater immunological cross-reactivity with bacterial ribosomal In E. coli and , as well as in the cyanobacte- proteins than those encoded in nuclear genes (510). The rium Synechocystis sp., the genes encoding Li, L10, Lll, and chloroplast-encoded proteins also show somewhat greater L12 are clustered (539, 564). The rpll, rplll, and rp112 genes sequence identity to their counterparts fromE. coli than do the also form a cluster in the cyanelle genome, but rpllO is nucleus-encoded ones (Table 3). Subramanian et al. (608) apparently missing (32). The rpoB and rpoC genes, which are made the interesting point that of 15 ribosomal proteins that part of the same cluster in E. coli, were found elsewhere in the can be individually eliminated by mutation in E. coli without Synechocystis genome and in the cyanelle genome. None of total loss ofviability (103, 104), the equivalent of only one, L33, these four ribosomal protein genes has been found in any land is chloroplast encoded in land plants. They suggest that plant chloroplast genome, but rpl12 is now known to be a location of particular chloroplast ribosomal protein genes to chloroplast gene in Euglena gracilis, located some distance the nuclear or chloroplast genome may be related to the from the rpoB and rpoC genes (243). essential roles of these proteins in ribosome assembly or function. In the following section we discuss each ribosomal protein in Correspondence of Chloroplast Ribosomal Proteins to turn, briefly describing what is known about its function and Bacterial Ribosomal Proteins structure in bacteria and indicating the chloroplast equivalents that have been identified. Table 3 provides a summary of Of the 54 ribosomal proteins that constitute the E. coli references for sequence information to complement this text. ribosome, the chloroplast equivalents of 44 have been identi- fied by sequencing of nuclear or chloroplast genes from one or Proteins of the Small Subunit more organisms (Table 3). Derived amino acid sequence identities for these proteins with their equivalents in E. coli are Protein SI is essential for mRNA binding in E. coli and may mostly in the range of 35 to 55%, with S12 showing consider- play an important role in initiation of translation of mRNAs ably greater conservation (Table 3). In addition, at least three that lack a Shine-Dalgarno sequence (604, 666). The gene VOL. 58, 1994 CHLOROPLAST RIBOSOMES AND PROTEIN SYNTHESIS 717

TABLE 3. Chloroplast and cyanobacterial ribosomal proteins that have been identified by gene or protein sequence E. N. Proteina Taxon GenBank accession no(s). Locationa colib tabacumb Reference(s) %S %I %S %I

Si Porphvra-Spra -a oeraepurpureaa C 49 26 59c 38c 517 Spinacia oleracea X66135, M82923 N 48 22 100 100 178

S2 Astasia longa X16004, X75651 C 52 31 56 33 566 Conopholis americana X64567 (partial) C 634 Cyanophora paradoxa C 66 48 65 49 598 Epifagus vitginiana X61798, M81884 C 58 38 92 85 435,712,714 Euglena gracilis X70810, Z11874 C 57 35 60 40 88, 243 Marchantia polymorpha X04465 C 63 45 83 73 659 Nicotiana tabacum Z00044 C 61 41 100 100 560, 561 Oryza sativa X15901 C 58 37 87 79 265 Pisum sativum X05917, X03912 C 61 42 94 87 97, 278 Porphyra purpurea C 66 47 70 50 517 Spinacia oleracea X05916 C 61 40 95 92 278 Spirulina platensis X53651 84 71 60 42 536 Triticum aestivum M35396 C 59 39 86 78 267 Zea mays X17318, X52270 C 59 39 87 79 282, 587, 588 S3 Chlamydomonas reinhardtii 5' X66250 C 44 25 49 25 172, 366 Chlamydomonas reinhardtii 3' X66250 C 57 36 56 34 172, 366 Cyanophora paradoxa M30487 C 72 51 67 47 423 Epifagus virginiana M81884 C 60 35 84 75 712,714 Euglena gracilis X70810, Z11874, M37463 C 69 51 58 35 88, 243 Gracilaria tenuistipitata M32638 C 64 46 65 43 294 Marchantia polymorpha X04465 C 67 43 76 62 186 Nicotiana tabacum Z00044 C 64 39 100 100 560, 561 Oryza sativa X15901 C 66 40 83 70 265 Porphyra purpurea C 68 51 69 46 517 Spinacia oleracea X13336 C 65 42 95 89 742 Zea mays Y00340, M31336 C 65 40 84 72 417 S4 Chlamydomonas reinhardtii C 59 38 64 48 277, 511 Chlorella ellipsoidea D10997 C 65 44 66 52 734 Cryptomonas 4) X51511 C 64 44 72 58 127 Cyanophora paradoxa C 63 41 74 59 32 Epifagus virginiana M81884 C 56 35 85 76 712, 714 Euglena gracilis X70810, Z11874, M22010 C 56 35 70 51 243 Marchantia polymorpha X04465 C 61 38 83 76 659 Nicotiana tabacum Z00044 C 58 36 100 100 560, 561 Oryza sativa X15901 C 61 38 89 80 265 Porphyra purpurea C 62 38 72 56 517 Spinacia oleracea M16878 C 58 38 95 92 22, 742 Zea mays X01608 C 59 38 88 79 610 S5 Cyanophora paradoxa M30487 C 64 42 423 Porphyra purpurea C 65 39 517 S6 Porphyra purpurea C 57 28 517 S7 Anacystis nidulans X17442 69 54 72 52 422 Astasia longa X14385, X75652 C 48 26 50 29 565 Chlamydomonas moewusii C 60 38 64 42 655 Chlamydomonas reinhardtii X53977 (partial; see text) C 56 37 63 44 509, 519 Cryptomonas 4) X52912 C 69 49 70 55 122 reflexa X72584 C 62 43 100 98 237 Cyanophora paradoxa X52497 C 67 49 71 53 326 Epifagus virginiana M81884 C 62 43 95 91 712, 714 Euglena gracilis X70810, X06254, X00480 C 62 40 62 41 243, 430 Glycine max X07675, X05013 C 63 43 99 97 672 Marchantia polymorpha X04465 C 66 43 88 78 659 Nicotiana tabacum Z00044, M19073 C 62 43 100 100 560,561 Oryza sativa X15901 C 60 43 92 85 265 Porphyra purpurea C 67 48 72 58 517 Spirodela oligorhiza X04508 (partial) C 498 Spirogyra maxima L07932 C 66 48 82 71 352 Spirulina platensis X15646 69 55 72 54 72 Continued on following page 718 HARRIS ET AL. MICROBIOL. REV.

TABLE 3-Continued E. N. Protein' Taxon GenBank accession no(s). Location" colib tabacumb Reference(s) %S %I %S %I Zea mays M17841 C 60 43 92 85 204 S8 Astasia longa X16004, X75651 C 60 34 64 38 566 Chlamydomonas reinhardtii C 65 44 69 53 277 Cyanophora paradoxa X16548 C 69 46 67 47 69 Cyanophora paradoxa M30487 C 68 46 67 47 423 Epifagus virginiana M81884 C 62 38 89 84 712, 714 Euglena gracilis X70810, Z11874 C 62 48 64 44 88, 243 Marchantia polymorpha X04465 C 67 46 78 59 186 Nicotiana tabacum Z00044 C 65 42 100 100 560, 561 Oenothera ammophila M60180 (partial) c 715 Oryza sativa X15901 C 62 41 86 76 265 Porphyra purpurea C 72 51 68 47 517 Spinacia oleracea X13336 C 53 37 89 79 742 Zea mays X06734 C 62 41 86 79 400 S9 Cryptomonas 4D X52912 (partial) c 122 Cyanophora paradoxa c 62 42 598 Euglena gracilis X70810 c 62 41 243 Porphyra purpurea c 64 44 517 S10 Cryptomonas 4D X52912 C 71 52 122 Cyanophora paradoxa X52143, M35206 C 71 50 68, 451, 452 Porphyra purpurea C 72 49 517 Sil Cyanophora paradoxa C 72 53 72 55 598 Epifagus virginiana M81884 C 71 54 90 83 712, 714 Euglena gracilis X70810, Z11874, M22010 C 58 38 69 44 243, 477 Marchantia polymorpha X04465 C 72 51 89 79 186, 659 Nicotiana tabacum Z00044 C 71 55 100 100 560, 561 Oryza sativa X15901 C 72 52 87 74 265 Pisum sativum X15645, X05029 C 72 54 93 84 504, 506 Porphyra purpurea C 72 55 74 55 517 Spinacia oleracea X03496 C 72 54 99 90 571 Zea mays M35831 C 72 52 88 73 401 S12 Anacystis nidulans X17442 82 74 89 81 422 Chlamydomonas reinhardtii M29284 C 76 68 84 78 364 Cryptomonas 4D X52912 C 82 73 90 84 122 Cuscuta reflea X72584 (partial) C 238 Cyanophora paradoxa X52497 C 83 77 88 84 326 Epifagus virginiana M81884 C 77 67 92 90 712, 714 Euglena gracilis X70810, X00480, X06254 C 82 69 85 73 243, 430 Glycine max X07675, X05013 C 78 70 98 98 672 Marchantia polymorpha X04465, X03661, X03698 C 81 71 94 92 187, 659 Nicotiana plumbaginifolia L12250, L12366 C 79 71 100 100 276 Nicotiana tabacum X03481, Z00044 C 79 71 - d d 183, 560, 561 Oryza sativa X15901 C 79 67 94 89 265 Pinus contorta L28807 (partial) C 89 Porphyra purpurea C 80 73 88 80 517 Spinacia oleracea (partial) C 540 Spirodela oligorhiza X04508 (partial) C 498 Spirogyra maxima L07931, L07932 C 81 73 90 87 352 Spinulina platensis X15646 82 74 88 81 72 Triticum aestivum X54484 (partial) C 218 Zea mays X60548, M17841, M17842 C 79 67 93 89 204, 687 S13 Cyanophora paradoxa C 71 54 598 Porphyra purpurea C 73 52 517

S14 Astasia longa X16004, X75651 C 62 46 68 55 566 Chlorella ellipsoidea D10997 C 65 50 67 52 734 Chlorella-like alga M74441, M81884 C 64 48 65 52 6 Cyanophora paradoxa C 65 46 71 54 598 Epifagus virginiana X61798 C 56 39 94 90 435, 712, 714 Euglena gracilis X70810, Z11874, X15240 C 61 42 61 46 88, 243 Continued on following page VOL. 58, 1994 CHLOROPLAST RIBOSOMES AND PROTEIN SYNTHESIS 719

TABLE 3-Continued E. N. Proteina Taxon GenBank accession no(s). Locationa col tabacumb Reference(s) %S %I %S %I Marchantia polymorpha X04465 C 58 44 82 75 659 Nicotiana tabacum Z00044 C 56 41 100 100 560, 561 Oryza sativa X15901, X13208 C 56 40 92 87 95, 265 Pisum sativum X05394 C 57 38 85 79 344 Porphyra purpurea C 56 40 70 56 517 Spinacia oleracea X04131 C 54 40 90 90 307 Zea mays Y00359, M16559 C 56 40 92 87 523,586 S15 Marchantia polymorpha X04465 C 53 36 72 58 312 Nicotiana tabacum Z00044 C 61 36 100 100 560, 561 Oryza sativa X15901 C 58 36 82 71 265 Secale cereale X14811 C 59 38 81 69 501 Zea mays X52614 C 60 41 86 77 170 S16 Cyanidium caldarium X62578 C 59 39 63 41 385 Hordeum vulgare X52765 C 62 38 90 86 556 Nicotiana tabacum Z00044, X03415 C 60 37 100 100 560, 561 Oryza sativa X15901 C 66 39 87 82 265 Porphyra purpurea C 58 42 71 51 517 Sinapis alba X13609 C 62 39 87 81 450 Solanum tuberosum Z11741 (partial) C 138 Zea mays X60823 C 63 37 90 84 293 S17 Arabidopsis thaliana J05215, Z11151 N 61 34 72e 55e 196, 640 Pisum sativum M31025 N 54 29 100 100 195, 196 Porphyra purpurea C 69 49 54e 39e 517 S18 Chlamydomonas reinhardtii C 53 34 63 41 348 Cyanophora paradoxa X17498 C 69 47 68 54 163 Epifagus viginiana M81884 C 58 34 86 81 712, 714 Euglena gracilis X70810, Z11874 C 61 37 70 48 134 Marchantia polymorpha X04465 C 58 38 88 74 186 Nicotiana tabacum Z00044 C 56 37 100 100 560, 561 Oryza sativa X15901 C 55 34 79 72 265 Porphyra purpurea C 69 48 72 51 517 Zea mays X56673 C 55 34 75 70 686 S19 Astasia longa X75653 C 63 44 66 43 211 Chlamydomonas reinhardtii C 78 67 81 62 277 Cyanophora paradoxa X17498 C 83 70 85 68 163 Epifagus virginiana M81884 C 65 47 84 83 712, 714 Euglena gracilis X70810, Z11874, M37463 C 71 57 72 54 88, 243 Glycine max X06429 C 75 54 97 92 584 Marchantia polymorpha X04465 C 77 62 88 80 186 Nicotiana debneyi C 75 56 100 100 745 Nicotiana tabacum Z00044, V00163 C 75 56 100 100 560, 561, 612 Oryza sativa X15901 C 65 45 84 70 265 Petunia hybrida M35955, M37322 (partial) C 3 Pisum sativum X59015 C 73 55 88 84 448 Porphyra purpurea C 78 62 76 60 517 Sinapis alba X17331 (partial) C 454 Spinacia oleracea X13336, X00797 C 76 57 97 92 635, 745 Zea mays Y00141 C 65 45 82 70 415 S20 Cyanophora paradoxa C 53 34 32 Porphyra purpurea C 51 33 517 S2lf Ll Cyanophora paradoxa C 65 45 32 Porphyra purpurea C 62 41 517 Spinacia oleracea N 64 43 300 Synechocystis strain PCC X73005 61 46 539 6803

L2 Astasia longa X75653 C 66 51 71 53 211 Continued on following page 720 HARRIS ET AL. MICROBIOL. REV.

TABLE 3-Continued E. N. Proteina Taxon GenBank accession no(s). Locationa colib tabacumb Reference(s) %S %I %S %I Chlamydomonas reinhardtii C 70 53 75 60 277 Cyanophora paradoxa X17498 C 68 52 76 61 163 Epifagus virginiana M81884 C 64 47 96 93 712, 714 Euglena gracilis X70810, Z11874, M37463 C 68 52 72 55 88, 243 Glycine max X06429 (partial) C 584 Marchantia polymorpha X04465 C 64 49 81 73 186 Nicotiana debneyi X00798 C 60 43 91 88 745 Nicotiana tabacum Z00044 C 66 48 100 100 560, 561 Oryza sativa X15901 C 64 49 93 90 265 Petunia hybrida M35944, M37322 (partial) C 3 Pisum sativum X59015 C 68 50 94 93 448 Porphyra purpurea C 67 50 73 59 517 Sinapis alba X65615 C 66 48 97 97 455 Spinacia oleracea X00797 C 63 44 90 85 745 Triticum aestivum (partial) C 46 Zea mays X53066, X12851, X62070 C 65 49 93 90 299 L3 Cyanophora paradoxa X17498 C 62 45 161 Porphyra purpurea C 63 45 517 L4 Porphyra purpurea C 60 38 517 L5 Astasia longa X16004, X14384, X75651 C 66 44 566 Chlamydomonas reinhardtii C 76 51 277 Cyanophora paradoxa X16548 C 76 51 69 Cyanophora paradoxa M30487 C 76 52 423 Euglena gracilis X70810, Z11874, X17051 C 69 44 88, 243 Porphyra purpurea C 74 54 517 L6 Cyanophora paradoxa X16548, M30487 C 63 38 69, 423 Porphyra purpurea C 58 41 517 L7 (see L12) L8 (see L10) L9 Arabidopsis thaliana Z11509, Z11129 N 53 32 82e 69e 640 Pisum sativum X14019 N 54 34 100 100 192 Porphyra purpurea C 61 30 55e 33e 517 Synechococcus sp. X63765 56 36 58e 34e 439 Synechocystis strain PCC D10716 57 34 62~ 38e 388 6803 L10 Synechocystis strain PCC X53178 53 26 539, 564 6803 Lll Arabidopsis thaliana N 63 51 93C 88C 543 Cyanophora paradoxa C 71 55 75C 63c 32 Porphyrapurpurea C 70 54 80c 65c 517 Spinacia oleracea X56615 N 63 52 100 100 579 Synechocystis strain PCC X73005 75 61 82C 69C 539 6803

L12 Arabidopsis thaliana X68046 (a) N 69 46 86 75 543, 689 Arabidopsis thaliana X68046 (b) N 64 40 71 59 543, 689 Arabidopsis thaliana X68046 (c) N 69 46 86 75 543, 689 Cyanophora paradoxa C 68 47 63 39 32 Euglena gracilis X70810 C 66 47 63 44 243 Nicotiana sylvestris S93166 N 69 48 99 99 354 Nicotiana tabacum X62368 N 70 48 99 99 155 Nicotiana tabacum X62339 N 70 49 100 100 155 Porphyra purpurea C 74 58 70 49 517 Secale cereale X68325 N 70 45 82 70 544 Secale cereale X68340 N 69 44 80 68 544 Spinacia oleracea J02849 N 75 53 89 78 201

Continued on following page VOL. 58, 1994 CHLOROPLAST RIBOSOMES AND PROTEIN SYNTHESIS 721

TABLE 3-Continued E. N. Proteina Taxon GenBank accession no(s). Locationa coli' tabacumb Reference(s) %S %I %S %I Synechocystis strain PCC X53178, X67516 78 62 70 45 539, 564 6803 L13 Porphyra purpurea C 66 51 69C 57C 517 Spinacia oleracea J04461 N 71 54 100 100 490 L14 Chlamydomonas reinhardtii X14062 C 77 57 88 71 372 Cyanophora paradoxa M30487 C 80 58 89 69 423 Euglena gracilis X70810, Z11874 C 76 60 85 63 88, 243 Marchantia polymorpha X04465 C 83 58 94 80 186 Nicotiana tabacum Z00044 C 80 55 100 100 560, 561 Oenothera ammophila M60179, M60180 (partial) C 715 Oryza sativa X15901 C 80 54 93 85 265 Porphyra purpurea C 80 60 85 67 517 Spinacia oleracea X13336 C 82 58 94 88 742 Vigna unguiculata M80799 (partial) C 8 Zea mays X06734 C 80 53 92 81 400 L15 Arabidopsis thaliana Z11507, Z11508 N 61 41 76e 68e 640 Pisum sativum Z11510 N 63 44 100 100 640 L16 Chlamydomonas reinhardtii M13931 C 79 57 82 69 373 Cyanophora paradoxa M30487 C 76 54 88 70 423 Epifagus virginiana M81884 C 76 50 90 81 712, 714 Euglena gracilis X70810, Z11874 C 73 53 77 65 88, 243 Gracilaria tenuistipitata M32638 C 72 60 77 65 294 Marchantia polymorpha X04465 C 75 56 90 79 186 Nicotiana tabacum Z00044 C 78 56 100 100 560, 561 Oenothera ammophila M60179 (partial) C 715 0ryza sativa X15901 C 76 54 90 86 265 Porphyra purpurea C 72 55 81 68 517 Spinacia oleracea X13336 C 76 53 96 90 742 Spirodela oligorhiza X03834 C 75 54 90 85 497 Vigna unguiculata M80799 (partial) C 8 Zea mays Y00375, X06734 (partial) C 400, 418 L17f L18 Cyanophora paradoxa M30487 C 62 49 423 Porphyra purpurea C 65 45 517 L19 Cyanophora paradoxa C 69 46 598 Porphyra purpurea C 69 47 517 Synechocystis strain PCC X72627 72 56 542 6803 L20 Astasia longa X75653 C 56 33 55 27 211 Chlamydomonas reinhardtii X62566 C 66 46 68 44 736 Cyanophora paradoxa X17063 C 72 53 71 52 69 Epifagus virginiana M81884 C 60 41 81 77 712,714 Euglena gracilis X70810, Z11874, Y00143 C 55 29 54 31 243, 396 Glycine max X07676 (partial) C 673 Marchantia polymorpha X04465 C 64 45 76 57 186 Nicotiana tabacum Z00044 C 63 41 100 100 560, 561 Oryza sativa X15901 C 59 41 78 67 265 Porphyra purpurea C 69 47 64 46 517 Zea mays X60548 C 58 41 81 71 687 L21 Cyanidium caldarium C 54 26 54C 32C 385 Cyanophora paradoxa C 58 31 57C 30C 598 Marchantia polymorpha X04465 C 54 29 55C 30c 312 Porphyra purpurea C 62 33 52C 32C 517 Spinacia oleracea M57413, M64682 N 60 32 100 100 340, 578 L22 Astasia longa X75653 C 53 31 60 34 211 Cyanophora paradoxa M30487, X17498 C 62 44 70 47 163, 423 Continued on following page 722 HARRIS ET AL. MICROBIOL. REV.

TABLE 3-Continued E. N. Proteina Taxon GenBank accession no(s). Locationa colib tabacumb Reference(s) %S %I %S %I Euglena gracilis X70810, Z11874, M37463 C 59 38 52 42 88, 243 Gracilaria tenuistipitata M32638 C 59 40 64 42 295 Marchantia polymorpha X04465 C 59 41 68 54 186 Nicotiana tabacum Z00044 C 54 36 100 100 560, 561 Oryza sativa X15901 C 53 32 70 54 265 Pelargonium zonale M60953 C 50 35 78 67 193 Pisum sativum M60951, M60952 N 58 43 72 53 193 Porphyra purpurea C 66 49 66 48 517 Zea mays Y00329 C 47 28 66 52 416 L23 Arabidopsis thaliana X66414 C 50 25 97 95 374 Astasia longa X75653 C 38 18 42 21 211 Chlamydomonas reinhardtii C 53 31 55 39 277 Euglena gracilis X70810, Z11874, M37463 C 42 17 53 32 88, 243 Marchantia polymorpha X04465 C 46 25 77 55 186 Nicotiana tabacum Z00044 C 48 23 100 100 560, 561 Oryza sativa X15901 C 51 26 94 84 265 Porphyra purpurea C 46 28 63 36 517 Sinapis alba X65615 C 50 25 98 96 455 Spinacia oleracea X07462 (pseudogene?) C 635 Triticum aestivum X12850 C 51 26 94 83 46 Zea mays X07864 C 51 26 93 84 419 L24 Nicotiana tabacum N 64 38 100 100 153 Pisum sativum X14020 N 56 34 86 78 192 Spinacia oleracea M58522 N 58 34 76 61 75 Porphyra purpurea C 51 33 61 49 517 L25f L27 Calyptrosphaera sphaeroidea D26097 (partial) C 185 Chlamydomonas reinhardtii N-terminal amino acid sequence only N 363 Chrysochromulina alifera D26096 (partial) C 185 Chrysochromulina hirta D26099 (partial) C 185 Cyanidium caldarium D26098 (partial) C 185 Nicotiana tabacum M75731 N 73 59 100 100 154 Pleurochrysis carterae D26100 C 77 58 77 61 185 Pleurochrysis haptonemofera D26102 (partial) C 185 Porphyra purpurea C 74 61 69 57 517 Porphyridium cruentum D26101 (partial) C 185 L28 Nicotiana tabacum X68078 N 57 36 731 L29 Porphyra purpurea C 51 24 517 L30f L31 Porphyra purpurea C 60 37 517

L32 Astasia longa X16004, X75651 C 56 38 55 45 568 Brassica rapa Z26332 C 48 15 85 83 582 Euglena gracilis X70810 C 48 30 50 35 243 Lycopersicon esculentum D17805 (partial) C 668 Marchantia polymorpha X04465 C 43 18 74 61 186 Nicotiana tabacum Z00044 (as ORF55) C 46 18 100 100 733 Oryza sativa X15901 C 47 22 67 56 265 Porphyra purpurea C 44 20 57 48 517 Vicia faba X51471 C 52 26 83 68 256 Zea mays X64099 C 43 18 74 61 688

L33 Cyanophora paradoxa X17498 C 60 42 78 62 163 Epifagus virginiana M81884 C 52 37 93 85 712,714 Marchantia polymorpha X04465 C 56 44 80 71 186 Nicotiana tabacum Z00044 C 52 37 100 100 560, 561 Oryza sativa X15901 C 60 42 82 73 265 Porphyra purpurea C 59 43 67 50 517

Continued on following page VOL. 58, 1994 CHLOROPLAST RIBOSOMES AND PROTEIN SYNTHESIS 723

TABLE 3. Chloroplast and cyanobacterial ribosomal proteins that have been identified by gene or protein sequence E. N. Proteina Taxon GenBank accession no(s). Locationa colib tabacumb Reference(s) %S %I %S %I Zea mays X56673 C 57 38 81 75 686 L34 Cyanophora paradoxa C 49 40 598 Porphyra purpurea C 60 49 517 L35 Cyanophora paradoxa X17063 C 55 38 61C 45c 69 Porphyra purpurea C 52 36 67C 50c 517 Spinacia oleracea M60449 N 64 42 100 100 577 L36 Astasia longa X16004, X75651 C 68 46 73 57 566 Cryptomonas 'F X62348 (partial) C 124 Cyanophora paradoxa C 78 62 92 76 598 Epifagus virginiana M81884 C 81 57 95 92 712, 714 Euglena gracilis X70810, Z11874 C 73 51 84 65 88, 243 Marchantia polymorpha X04465 C 86 62 95 86 186 Nicotiana tabacum Z00044 C 86 62 100 100 560, 561 Oryza sativa X15901 C 89 68 95 92 265 Pisum sativum Y00468, X15645 C 84 62 97 86 505, 506 Porphyra purpurea C 81 59 95 70 517 Spinacia oleracea X03496 C 84 62 97 95 571 Zea mays M35956 C 89 68 95 92 402

CS-S5, PSrp-1, "S22" or "S30"g Spinacia oleracea X59270, X15344 N 28, 741 Spinacia oleracea M55322 N 290 "S31" or SCS239" Spinacia oleracea 541, 674 PsCL189 Pisum sativum X14021 N 192 "L40999 Spinacia oleracea M58523 N 75 PsCL25M Pisum sativum X14022 N 192 a Proteins Si through S21 and LI through 136 are named by reference to similar sequences in E. coli, and the location of the gene encoding them (N, nuclear; C, chloroplast or cyanelle) is given for all eukaryotic species. A few additional chloroplast ribosomal proteins with no obvious similarity to E. coli proteins have been identified and appear at the end of the table. b The percent similarity (%S) and percent identity (%I) to the E. coli and tobacco proteins or other reference land plant proteins were calculated by the gap routine of the Genetics Computer Group sequence analysis package. c Tobacco sequence not available; spinach used instead for comparison. d The complete tobacco genome sequence (Z00044) has a stop codon in the terminal exon encoding the S12 protein, whereas the sequence by Fromm et al. (183) (X03481) shows a full-length protein comparable to that from other chloroplast genes. The Swissprot sequence (P06309) omits the terminal residue (Tyr) of the first exon of this protein. The composite sequence with these corrections made is identical to that for N. plumbaginifolia and was used for the comparisons given here. I Tobacco sequence not available; pea used instead for comparison. f No equivalent found so far in chloroplasts. g Proteins for which no equivalent appears to exist in E. coli. h Small, basic protein found in spinach ribosome preparations.

encoding this protein is absent from the completely sequenced In E. coli the S2 protein interacts primarily with the 3' chloroplast genomes of tobacco, rice, Marchantia polymorpha, domain of the 16S rRNA in the 960 loop region and can be and Euglena gracilis (243, 558) but appears in the Porphyra cross-linked to proteins S3, S5, and S8 (500, 595, 599). The rps2 purpurea chloroplast genome (514). A nuclear gene encoding gene encoding a ribosomal protein homologous to E. coli S2 this protein has been identified in spinach (178, 179) and has been found in chloroplast genomes from diverse species shown to have a light-independent, leaf-specific pattern of (Table 3), typically mapping between the rpoB and rpoC genes expression under the control of a negative nuclear factor, SlF, encoding subunits of RNA polymerase and the atpI and atpH that down-regulates its promoter (740). Hahn et al. (240) genes encoding ATP synthase subunits. The deduced amino reported that monoclonal antisera to E. coli Si reacted with a sequences of S2 proteins from land plants are highly conserved chloroplast protein of spinach. Polyclonal antisera to two (Table 3), and those of three monocots (wheat, rice, and chloroplast-synthesized ribosomal proteins of C. reinhardtii maize) are nearly identical to one another. A nuclear DNA ("S-7" and "S-11" [547]) reacted with E. coli Si (510), as did sequence in spinach with substantial homology to a portion of antisera to mixed chloroplast ribosomal proteins of spinach the chloroplast rps2 gene is thought to be an example of (119). Subramanian et al. (608) also suggested the presence of "promiscuous DNA," i.e., a DNA sequence found in more a chloroplast Si homolog in maize on the basis of affinity- than one genetic compartment (85). binding experiments with a matrix-bound poly(U) column. Protein S3, like S2, interacts with nucleotides in the 960 and 724 HARRIS ET AL. MICROBIOLE REV.

1050/1200 regions of E. coli 16S rRNA (465) and appears from ending with rps8 (Fig. 5) but is separated from this operon by immunoelectron microscopy studies of the 70S ribosome to exon 1 of the psaA gene and two tRNA genes, tmM and trnG reside near the site to which the polypeptide release factor (277). In Euglena gracilis, rps4 is transcribed together with RF-2 binds (632). The rps3 gene encoding the equivalent rpsll (242, 597). In the alga Cryptomonas 'F, rps4 is close to protein has been sequenced from several chloroplast genomes rbcL on the same strand and is flanked by tRNAArg on the (Table 3), where it has been found between rp122 and rp116, as opposite strand (127), whereas in Cyanophora paradoxa, is true for the corresponding genes in the S10 operon of E. coli tRNA et and tRNAG'Y are adjacent to rps4 but on the (Fig. 5). In most algae and plants the rps3 gene is uninter- opposite strand (32, 598). rupted, but in Euglena gracilis it contains two introns, one (102 In E. coli, the S5 protein is part of the recognition complex nt) belonging to the group III class (as defined by Christopher (466) and is the first protein of the small subunit whose crystal and Hallick [87]; also see reference 86) and the other (409 bp) structure has been determined (from B. stearothermophilus being a "twintron" consisting of a 311-nucleotide group II [507, 508]). The protein appears to be a somewhat elongated intron within a 98-nt group III intron (92). Splicing proceeds molecule with two distinct domains. Mutations affecting amino sequentially, with the internal 311-nt intron being excised first. acids 20 to 22 ofE. coli S5 can confer spectinomycin resistance, In the chloroplast genome of C. reinhardtii, there is no gene whereas mutations at amino acids 104 and 112 have a ram equivalent to rps3 in the expected location between rp122 and phenotype and suppress streptomycin dependence mutations rp116 (277). However, Fong and Surzycki (172) found a long in protein S12 (508). Some of the latter class of mutants are open reading frame between the rpoB and rpoC genes, whose also neamine resistant (721). These two conserved regions of 5' and 3' ends would encode a protein with substantial the S5 protein are thought to be the sites of its interaction with homology to S3. The central portion of the predicted product rRNA (508). Genes encoding a protein with homology to E. of this open reading frame has no homology to S3, however, coli S5 have been sequenced from the cyanelle genome of and the DNA sequence does not contain recognizable splice Cyanophora paradoxa (368, 423) and from the P. purpurea junctions that would suggest that this region is in fact an intron. chloroplast genome (517). No equivalent gene has been found Liu et al. (366) found that this open reading frame is also in any land plant chloroplast genome, however, nor does it present in several other Chlamydomonas species. After trans- appear in the chloroplast genome of Euglena gracilis. formation of C. reinhardtii cells with a construct containing this The S6 ribosomal protein of E. coli is implicated in mRNA open reading frame interrupted by the bacterial aad antibiotic and tRNA binding and in termination (465, 632), and it resistance gene, the only resistant cells recovered were hetero- appears to be a component of the platform region of the 30S plasmic for the interrupted and native forms of the gene. In particle (432, 622). A plastid equivalent of S6 is known so far contrast to transformants in which the same construct was only from Porphyra purpurea (517). inserted into other regions of the genome, no homoplasmic In E. coli, protein S7 interacts with several clusters of cells containing only the interrupted gene could be obtained, nucleotides in the 3' domain of 16S rRNA, in proximity to S9, strongly suggesting that this gene is not only functional but also S1O, and S19 (57, 136, 465, 499), and is one of the initiating essential to cell growth. No single transcript spanning the proteins of 30S assembly (255). Binding of S7 to 16S riRNA is whole gene could be detected, however, and the gene product a prerequisite to assembly of S9 and S19. As discussed above, has not been identified (366). the gene encoding S7 is transcribed together with that for S12 In E. coli, S4 is one of the primary rRNA-binding proteins in bacteria and in the chloroplast genomes of most plants and that initiate assembly of the 30S subunit (255, 465) and is algae examined (Fig. 5), the principal exceptions so far being associated with the 5' domain of 16S rRNA, at a junction of Chlamydomonas species. In C. reinhardtii, the protein encoded several helices. Together with S5 and S12, S4 participates in a by rps7 corresponds immunologically to the protein that region designated by Oakes et al. (466) as the recognition Schmidt et al. (547) identified as "S-20" (509). Although complex on the basis of its demonstrated involvement in derived amino acid sequence identity between chloroplasts and codon-anticodon recognition and translational accuracy. This bacteria is lower for S7 than for S12 (Table 3), antibodies toE. region involves the 530 loop, the 900 loop region, and the 5' coli S7 do cross-react with a corresponding small-subunit end of the 16S molecule which pairs with the region around protein from spinach chloroplast ribosomes (18). residue 912 (Fig. 3). Homologs of all three of these proteins In E. coli, S8 is an RNA-binding protein that is essential have been identified in yeast cytoplasmic ribosomes and ap- early in assembly of the 30S subunit and interacts with a highly pear to have similar functions (4). Mutations in the gene conserved site in the central domain of 16S rRNA, designated encoding S4 in E. coli suppress streptomycin dependence by Oakes et al. (466) as the platform ring (also see references mutations in the gene for S12 and increase translational 141, 437, 465, and 622). It is associated with proteins S15 and ambiguity (ram mutants [7, 189, 335, 336, 475). The chloroplast S17 (57). It also has a key role in translational regulation of the gene encoding ribosomal protein S4 has been sequenced from spc operon in E. coli (719). S8 is moderately conserved a number of plants and algae (Table 3) and shows a high phylogenetically and can be identified with equivalents in degree of conservation in its first 25 amino acid residues and in eukaryotic ribosomes (410, 708). The rps8 gene is chloroplast a large block of approximately 120 residues in the central encoded (Table 3) and is one of at least three genes of the portion of the protein. The C. reinhardtii S4 protein is some- bacterial spc operon that remain linked in chloroplast genomes what longer than all others examined so far, having two (Fig. 5). Most plastid S8 proteins have a central 4- to 7-amino- internal insertions and a 22-amino-acid C-terminal extension acid insertion compared with the E. coli protein, followed by a (511). highly conserved C-terminal region. In land plants, rps4 appears to be transcribed singly under Protein S9 interacts with S7 and S19 in the 3' domain of the control of its own promoter and is not part of an operon with E. coli ribosome (57, 499). Bartsch (18) obtained cross-reac- other ribosomal protein genes (608). In tobacco, rice, and tivity of antibody to E. coli S9 with a spinach chloroplast Marchantia polymorpha, the rps4 gene is in the large single- ribosomal protein, but the gene encoding this protein has not copy region and is preceded by tRNAThr on the same strand been found in any of the land plant chloroplast genomes so far and followed by tRNAser on the opposite strand. In C. sequenced and is presumed to be nucleus encoded. However, reinhardtii, rps4 follows the large ribosomal protein operon an rps9 gene does appear in the chloroplast genomes of VOL. 58, 1994 CHLOROPLAST RIBOSOMES AND PROTEIN SYNTHESIS 725

Euglena (243), Porphyra (517), and Cryptomonas (122) species the 16S rRNA and is probably close to S4 in the assembled 30S and in the cyanelle genome (598). Overall, the C-terminal subunit (594). Montesano-Roditis et al. (432) have localized portions of the S9 proteins appear more highly conserved than S16 to the 30S body near its junction with the platform, on the the N-terminal portions. surface facing the 50S particle. In various angiosperms, S16 is S10 is a late-assembly ribosomal protein of the 3' domain in encoded by a chloroplast gene (Table 3), but the rps16 gene is E. coli 16S rRNA (465). A gene encoding a homolog ofS10 has absent from the Marchantia chloroplast genome (471). It is been found in the cyanelle genome (68, 451, 452), where it found, however, in the plastid genome of the red alga Cya- maps downstream of the petFI gene. Sequences encoding an nidium caldarium (385). In tobacco, the rpsl6 gene has an S10 protein have also been found in Cryptomonas D (122) and 860-bp intron with boundary sequences similar to the introns in in Porphyra purpurea downstream of the tufA gene (517). rpsl2, rp12, and several tobacco tRNA genes (559). The rpsl6 In E. coli, Sl1 interacts with S6 and S18 in the platform genes of mustard (450), barley (556), and maize (293) also region of the 30S subunit (466). S11 is highly conserved contain introns, but that of Cyanidium caldarium is uninter- phylogenetically, with homologs also identified in eukaryotic rupted (385). ribosomes (401, 708). There is some variability in length S17 is one of the primary assembly proteins in E. coli and among Sli proteins, localized to the N-terminal regions. In binds to 16S rRNA in the 5' domain (213, 255, 465, 594). It is chloroplast genomes of land plants, the rpsll gene is part of one of only three chloroplast ribosomal proteins of the small the large ribosomal protein operon that terminates in rpoA subunit to date for which a nucleus-encoded gene has been (Fig. 5). In Euglena gracilis, in which it is in a separate operon cloned and sequenced (Table 3) (606). This protein contains a with rps4, the rpsll gene contains two group III introns (597). highly conserved region which can be identified in both S12 appears to be the most highly conserved of all the small bacterial (S17) and eukaryotic (Sli) ribosomal proteins. Com- subunit proteins. The C. reinhardtii S12 protein is sufficiently parison of the deduced amino acid sequences of the equivalent similar to its bacterial counterpart that the rpsl2 gene from this chloroplast S17 and cytosolic Sli ribosomal proteins from alga can be expressed in E. coli cells and the resulting protein Arabidopsis thaliana with E. coli S17 supports the notion that can assemble and function in the E. coli ribosome (364). In E. chloroplast S17 is derived from a prokaryotic coli, S12 interacts with two regions of the 16S rRNA, the 530 and not from duplication of the eukaryotic S11 gene (196). The loop and 900 stem-loop. Mutations affecting either the S12 presence of an rpsl7 gene in the cyanelle genome of Cyano- protein or the 16S rRNA regions with which it associates can phora paradoxa (368) and in the chloroplast genome of Por- confer streptomycin resistance in E. coli by reducing misread- phyra purpurea (514) is consistent with this hypothesis. ing induced by the drug (Table 2). Streptomycin resistance Proteins S18 and S6 assemble coordinately in the bacterial mutations have also been found at evolutionarily conserved ribosome to form part of the platform ring in the central sites in the S12 proteins or 16S rRNA of Chlamydomonas domain (432, 466, 622). The chloroplast gene encoding S18 has species, Euglena gracilis, and tobacco, and streptomycin depen- been found to be part of an operon with rp133 in the completely dence mutations affecting the S12 protein have been found in sequenced chloroplast genomes of tobacco, rice, and Mar- bacteria and in C. reinhardtii (Table 2). Streptomycin-depen- chantia polymorpha, as well as in Cyanophora paradoxa, but it dent E. coli mutants exhibit hyperaccurate proofreading and is absent from this operon in Euglena gracilis (Fig. 5). The reduced efficiency of binding of EF-Tu (25, 102, 150). angiosperm S18 proteins have N-terminal extensions com- In E. coli, S13 appears to be located at the head of the 30S pared with E. coli, Marchantia polymorpha, and Cyanophora subunit, near the center of the surface that faces the 50S paradoxa, containing various numbers of repeats of a hydro- subunit (432). It has been reported to cross-link to S7 and S19 philic heptapeptide (686). A C-terminal extension found in the in the 3' domain of the 16S rRNA (599). Genes encoding a S18 proteins of rice and maize is missing from this protein in cognate protein have been identified in the cyanelle genome tobacco (561, 686). (598) and in the Porphyra chloroplast genome (517) but have The S19 protein of E. coli interacts with proteins S7, S9, and so far not been reported to occur in other algae or land plants. S14 and with several helices in the 3' domain of the 16S rRNA The late-assembly ribosomal protein S14 interacts with the molecule (57). The gene encoding S19 was the first chloro- 3' domain of 16S rRNA in E. coli (465). The chloroplast- plast-encoded ribosomal protein gene to be identified and encoded Euglena rpsl4 gene is part of the large ribosomal sequenced (612, 745). In plants with the "typical" chloroplast protein cluster as it is in E. coli, downstream from rp136 and a inverted-repeat structure, the rpsl9 gene and the adjacent rp12 tRNAIle gene (457), whereas in other chloroplast genomes it is and rp122 genes are located at or near the boundary between found outside this cluster. In land plants, the rpsl4 gene is the inverted repeat and the large single-copy region. In to- located in the chloroplast genome downstream from the psaA bacco (560) and Marchantia polymorpha (186), rpsl9 is entirely andpsaB genes encoding reaction center proteins of photosys- within the large single-copy region but near the inverted-repeat tem 1(81, 82, 307, 496, 560). In Cyanophora paradoxa, the rpsl4 junction, whereas in rice (433) the whole gene is within the gene is upstream of open reading frame ORF512 andpsaA and inverted repeat. Zurawski et al. (746) found that the first 48 is transcribed divergently from these two genes (598). In codons of rpsl9 in spinach were in the inverted repeat, with 44 Porphyra purpurea, rpsl4 is flanked by petF and petG (514). codons homologous to the 3' end of the E. coli gene being S15 is an early-assembly protein in E. coli that interacts with present only on one side of the large single-copy region. the central domain of the 16S rRNA, together with S6, S8, and Thomas et al. (635, 636) showed that this complete copy of the S18 in the platform ring (57, 151, 438, 465, 466, 622). In gene was expressed, whereas the rpsl9' sequences beginning in tobacco and liverwort, the rpsl5 gene is in the small single-copy the other side of the inverted repeat and extending for 66 region of the chloroplast genome (312, 560), whereas in three codons into the adjoining unique sequence region (745) were monocots (rice [265], rye [501], and maize [170]) it is in the not transcribed. The rpsl9 gene also straddles the boundary of inverted repeat, very close to the boundary with the small the inverted repeat in Spirodela oligorhiza (494) and in mustard single-copy region. This gene is missing from the chloroplast (454). genomes of Euglena gracilis (243) and Porphyra purpurea (517) S20, which was also identified in early ribosome studies in E. and from the cyanelle genome (32, 598). coli as L26, is a primary RNA-binding protein that interacts In E. coli, S16 is a protein associated with the 5' domain of with the 5' domain of the 16S rRNA (255,594). Deletion of the 726 HARRIS ET AL. MICROBIOL. REV.

rpsT gene encoding S20 in E. coli results in increased misread- families and documented six independent losses of this intron ing of all three nonsense codons and a deficiency in assembly among dicotyledons. of 30S and 50S subunits to form 70S monomers (534). An rps2O The L3 and L4 proteins of E. coli both bind to the 23S rRNA gene has been identified in the cyanelle genome (32) and in the molecule and have been identified with analogous proteins in Porphyra chloroplast (517). archaebacterial and eukaryotic ribosomes (215, 708). Genes E. coli S21 interacts with the central domain in the platform encoding an L3 protein have been sequenced from the Cyano- ring (466). No equivalent protein has been identified so far in phora cyanelle genome (161) and from the Porphyra chloro- the chloroplast ribosome. plast genome (517). Neither an rp13 nor an rpl4 gene has been found in the completely sequenced chloroplast genomes of Proteins of the Large Subunit tobacco, rice, Marchantia polymorpha, or Euglena gracilis Bartsch (18) found a cross-reaction between antibody toE. coli In E. coli, ribosomal protein Li forms a prominent ridge on L3 and a spinach chloroplast ribosomal protein which has not the large subunit (177, 466,599) and has been demonstrated to been further characterized. bind to nt 2100 to 2200 on theE. coli 23S rRNA, a region that Genes encoding a protein corresponding to the 5S RNA- shows a high degree of conservation among chloroplast 23S binding protein LS of E. coli have been found in the Euglena, sequences. The gene encoding this protein is present in the C. reinhardtii, Porphyra, Astasia, and Cyanophora plastid ge- cyanelle genome (32) and in the Porphyra chloroplast genome nomes (Table 3). However, no equivalent gene has been found (517) but is absent from the completely sequenced plastid in any land plant chloroplast genome. Antibody to Chlamydo- genomes of land plants. Antibodies to E. coli Ll cross-reacted monas chloroplast ribosomal protein "L-13" (547) reacts with with a spinach chloroplast ribosomal protein (8). cDNAs E. coli LS and with a ribosomal protein ofAnabaena sp. (510). encoding chloroplast Li have been cloned from the nuclear A weak reaction was also seen to a spinach protein ("LIO"), genomes of pea, spinach, and Arabidopsis thaliana, and the whose site of synthesis is uncertain (121). The Chlamydomonas nuclear gene has been isolated and characterized from Arabi- "L-13" protein is known to be synthesized in the chloroplast dopsis thaliana (300). (547), suggesting that this is the product of the chloroplast- In E. coli, the L2 protein binds to domain IV of the 23S encoded rplS gene sequenced by Huang and Liu (277). rRNA molecule, and cross-links specifically to nt 1818 to 1823 TheE. coli L6 protein binds to domain VI of 23S rRNA (90). (744), in a stem-loop structure that is part of the peptidyltrans- Genes encoding an equivalent protein have been found in the ferase center and is conserved in chloroplast 23S rRNAs. cyanelle genome of Cyanophora paradoxa (69) and the Por- Site-directed mutagenesis of a conserved region in the E. coli phyra chloroplast genome (514). L2 protein outside the 23S binding site has been used to The protein originally identified as L7 in E. coli is in fact the produce temperature-sensitive mutants that are impaired in aminoacetylated form of L12, and L8 is a complex of L7/L12 assembly of the 50S subunit (526). The L2 protein is encoded and L10 (358). in the chloroplast genomes of all land plants and algae so far Protein L9 of E. coli is an elongated protein with distinct examined (Table 3). The L2 protein itself is moderately terminal domains which is associated with the protuberance conserved, and its equivalent has been identified in eukaryotic formed by protein Li and the region of the 23S rRNA to which ribosomes (708). The C. reinhardtii protein "L-1," which is it binds (57, 213, 266). Genes encoding a protein equivalent to synthesized in the chloroplast (547), appears to be encoded by E. coli L9 have been sequenced from the nuclear genomes of the rpl2 gene since "L-1" antibodies cross-react with E. coli L2 pea (192) and Arabidopsis thaliana (640) and from the Por- (510). Kamp et al. (292) showed that the N-terminal amino phyra chloroplast genome (517). The E. coli L9 protein cross- acid of the L2 protein in spinach is N-methylalanine, the first reacts slightly with antibody to the acidic chloroplast ribosomal demonstration of methylation of a chloroplast ribosomal pro- protein "L-30" from C. reinhardtii (510). tein. Several ribosomal proteins ofE. coli are methylated, but E. coli protein L10 (L8) forms the base of the ribosomal L2 is not among these. The maize rpl2 gene begins with an stalk in a pentameric complex with two dimers of L7/L12 and, ACG codon, which is edited to AUG at the transcript level like L7/L12, appears to be a universal constituent of eubacte- (321). The 3'-terminal ends of the deduced L2 amino acid rial, eukaryotic, and archaebacterial ribosomes (708). A cya- sequences for spinach and Nicotiana debneyi published by nobacterial gene encoding this protein has been found (539, Zurawski et al. (745) appear to lack homology to the corre- 564) but no chloroplast equivalent has yet been identified. sponding regions from other chloroplast and bacterial L2 Presumably, chloroplast L10 is encoded by a nuclear gene in proteins. However, if a single-base insertion is made after the plants. amino acid 226 of the spinach gene (changing the sequence L1i is an early-assembly protein that constitutes part of the CCC ACG GGG GTG GTG ....[Pro Thr Gly Val Val.1..].. to GTPase center in theE. coli ribosome (528) and also has been CCN CAC GGG GGT GGT ....[Pro His Gly Gly Gly....], the identified in eubacterial, eukaryotic, and archaebacterial ribo- reading frame is shifted to specify 45 additional amino acids somes (708). Nuclear genes encoding a chloroplast L1 ho- that resemble the consensus sequence much more closely. The molog have been cloned from spinach andArabidopsis thaliana corresponding change in the N. debneyi sequence produces a C (543, 579, 606), while plastid genes have been identified in terminus identical to that of N. tabacum as determined by Porphyra purpurea (517) and Cyanophora paradoxa (32). InE. Shinozaki et al. (561). The rp12 genes of tobacco, spinach, rice, coli, L1i has the most extensive posttranslational modification and maize are located in the inverted-repeat region, but those (nine methyl groups) of all ribosomal proteins (708); the same of Marchantia polymorpha and C. reinhardtii are in single-copy modifications occur in the spinach chloroplast Lii in the DNA. The spinach, C. reinhardtii, and Euglena genes are corresponding amino acid residues, located in conserved se- uninterrupted, whereas those of many other land plants con- quence contexts (607). Mutations in Bacillus megaterium and tain a single group II intron (133). The intron insertion sites B. subtilis conferring resistance to the antibiotic thiostrepton are identical in the Nicotiana, rice, and Marchantia genes, and cause the loss of Lii from the ribosomes (16, 101). Lli does the introns themselves have a high degree of nucleotide not bind thiostrepton itself in solution but enhances thiostrep- sequence identity. Downie etal. (133) determined the distri- ton binding to 23S rRNA (102, 639). Bacterial thiostrepton- bution of the rpl2 intron in 390 species from 116 angiosperm resistant mutants with altered 23S rRNA have also been found VOL. 58, 1994 CHLOROPLAST RIBOSOMES AND PROTEIN SYNTHESIS 727

(149, 533). McElwain et al. (413) have isolated a thiostrepton- protease digestion, suggesting that it remained on the ribo- resistant mutant of C. reinhardtii whose chloroplast ribosomes some surface (203). are resistant to the drug in vitro. The large subunits of the L14 in E. coli is a late-assembly protein and does not bind mutant ribosomes lack a cytoplasmically synthesized protein directly to 23S rRNA (458). Antibody-binding studies suggest ("L-23") that, on the basis of size and immunological criteria, that in B. stearothermophilus this protein is located on the appears to be the equivalent of the E. coli L1i protein. In surface of the 50S subunit (599). Genes encoding proteins with pulse-labeling experiments, this Chlamydomonas mutant syn- a high degree of homology to E. coli L14 have been found in thesizes small amounts of protein "L-23," but the protein fails chloroplast genomes of all plants and algae examined, except to assemble into chloroplast ribosomes. The mutation shows in the Epifagus virginiana, in which a pseudo- Mendelian inheritance, suggesting that rplll is a nuclear gene gene is present instead (714) (Table 3). in C. reinhardtii. In E. coli, both L15 and L16 are late-assembly proteins that The acidic protein L7/L12 is one of the most intensively are associated with the peptidyltransferase center but seem to studied ribosomal components (57, 474, 708). Four L12 mol- be nonessential for ribosome function. Fully active ribosomes ecules are present in each ribosome of E. coli, together with lacking both these proteins, as well as L30, can be reconstituted one L10 polypeptide, forming the stalk of the 50S subunit and in vitro by modifying the conditions of the reconstitution interacting with elongation factor EF-G which binds near the procedure (175). Nuclear genes encoding chloroplast ribo- base of the stalk. The homologous protein was identified in somal protein L15 have been reported from Arabidopsis thali- spinach chloroplasts by direct sequencing of tryptic peptides ana and pea (640), and no rpll5 sequences have been identified (19) and was predicted to have a tertiary structure similar to in any chloroplast genome to date (Table 3). The spinach that of its counterpart in E. coli (271, 345). cDNA clones chloroplast homolog of L15 is significantly larger than its encoding this protein have been isolated and sequenced from counterpart in E. coli, owing to extensions at the N-terminal the nuclear genomes of several land plants (Table 3), and the and probably also the C-terminal ends (291). gene has been cloned and characterized from Arabidopsis In contrast to L15, ribosomal protein L16 is encoded by a thaliana and spinach (689). In Arabidopsis thaliana (689), L12 chloroplast gene located in a conserved operon in all species is encoded by a multigene family with one silent and two examined (Table 3; Fig. 5). In land plants, the rpll6 gene is split functional genes, the functional genes both being closely linked into two exons (e.g., 498), whereas in Chlamydomonas rein- to cytosolic tRNA genes (this is the first such case identified for hardtii (373) and Gracilaria tenuistipitata (294), it is uninter- a chloroplast ribosomal protein [607]). However, L12 is en- rupted. The Euglena gene contains three introns (86, 93). coded by a chloroplast gene in Euglena gracilis and by a Antibodies to a chloroplast-encoded Chlamydomonas protein cyanelle gene in Cyanophora paradoxa. The derived amino acid ("L-17") cross-react with E. coli L16, with spinach "L24," and sequence of the cloned spinach gene includes three amino with an Anabaena protein that comigrates with E. coli L16 acids that were apparently overlooked in the primary sequence (510). of the corresponding protein published by Bartsch et al. (19). Protein L17 of E. coli has been shown to bind 23S rRNA Sibold and Subramanian (564) have compared the spinach and (358). A mitochondrial homolog has been identified in Sac- bacterial sequences with the L12 protein of the cyanobacte- charomyces cerevisiae (708), but no corresponding chloroplast rium Synechocystis sp. Like the E. coli protein, spinach L12 is protein has been found. However, proteins of similar charge present in multiple copies per ribosome, but it lacks the and size were seen on two-dimensional electrophoresis of N-terminal acetylation seen in the bacterial protein (19). ribosomal proteins from C. reinhardtii and Anabaena sp. (510). Giese and Subramanian (202) reported that the transit Antibodies to E. coli L17 were also observed to cross-react with peptide sequence of a spinach gene for L12 contains two ATG a chloroplast ribosomal protein from spinach (18). codons, each in a consensus initiation context, that would yield Protein L18, which is highly conserved in bacteria, binds to the same mature peptide after transport into chloroplast and 5S rRNA and is associated with the peptidyltransferase center N-terminal cleavage. Genes for L13, L35, and the novel (91). Genes encoding a ribosomal protein equivalent to E. coli protein Psrp-1 have similar duplicated ATGs. Experiments in L18 have been found in the cyanelle genome of Cyanophora which the 5' part of the L12 gene was fused to a reporter gene paradoxa (423) and in the Porphyra chloroplast genome (517). demonstrated that both codons can be used in vitro and in Spinach ribosomal protein "CS-L13" is a homolog of E. coli spinach protoplasts, with about 25% of initiations occurring at L22 (see below), with N- and C-terminal extensions that have the second codon. Such an arrangement may enhance transla- no sequence homology with the 5S-binding proteins of E. coli tional efficiency. However, Elhag et al. (155) found that both but show some structural similarity to L18 (651). L12 cDNAs from tobacco had only a single ATG, correspond- A gene encoding a protein equivalent to E. coli L19 has been ing to the first ATG of the spinach gene. found in the Porphyra chloroplast genome (514) and in the The L13 protein of E. coli interacts with the 5' domain of the cyanelle genome (598). Little is known about the function of 23S rRNA molecule, in proximity to L4, L21, L28, and L29 this protein in either bacterial or chloroplast ribosomes. (57). A nuclear cDNA clone encoding a chloroplast ribosomal An equivalent of the early-assembly, RNA-binding protein protein equivalent to E. coli L13 has been identified in spinach L20 of E. coli is encoded in chloroplast genomes of plants and (490, 609), and a chloroplast gene for this protein has been algae and in the cyanelle genome of Cyanophora paradoxa found in Porphyra purpurea (517). The spinach protein has (Table 3). 54% deduced amino acid identity with that of E. coli over the Protein L21 of E. coli can be cross-linked to the 5' domain 142 amino acid residues that can be aligned, but it is preceded of the 23S rRNA molecule and, together with L4, may have a by 52 residues at the N terminus with no homology to any second contact to the 23S molecule in the adjacent domain (57, known protein. Upstream of this sequence are 47 amino acids 481). The gene encoding L21 is chloroplast encoded in Mar- which appear to be a transit peptide. The chloroplast protein chantia polymorpha and in the red algae Cyanidium caldarium also has a C-terminal extension with no homology to E. coli and P. purpurea (Table 3) but is absent from the chloroplast L13. However, spinach L13 translated in E. coli from cDNA genomes of rice, tobacco, and Euglena gracilis. A spinach constructs was found to be incorporated into functional ribo- nuclear gene encoding L21 has been found to contain four somes (203). The N-terminal extension was removable by mild introns in its central region (340, 578). Two transcription start 728 HARRIS ET AL. MICROBIOL. REV. sites were identified, one which appears to be constitutive and L23 are found in ribosomes from eubacteria, organelles, the other which appears to be induced only in leaf tissue (340). archaebacteria, and cytoplasmic ribosomes of (513, The spinach L21 protein (formerly "CS-L7" [378]) is consid- 708). The equivalent cytoplasmic ribosomal proteins (called erably longer than its homologs from E. coli and Marchantia L25 proteins) have an extended amino terminus and a carboxy polymorpha, having extensions at both the N and C termini. terminus that resembles the archaebacterial L23 protein more The carboxyl-terminal extension contains seven Ala-Glu re- than the eubacterial one. peats, creating a region of high negative charge, and the Sequences with relatively low homology to the gene encod- protein as a whole is acidic, in contrast to E. coli L21, which is ing E. coli L23 have been found in the "S1O"-like operons in basic. However, this protein can be incorporated into E. coli chloroplast genomes of a number of plants (Table 3; Fig. 5). ribosomes assembled in vivo (71, 685). The spinach L21 While these rp123 sequences are in the same position as theE. protein shows greater homology to E. coli L21 than it does to coli gene for L23 in this operon, not all the chloroplast the chloroplast-encoded Marchantia protein, prompting Mar- sequences form continuous open reading frames and some tin et al. (408) to hypothesize that spinach L21 arose either by may be pseudogenes (45, 732, 746). The rp123 genes of spinach duplication of a nuclear gene for a corresponding protein of and four related dicots appear to have sustained a 14-bp the cytoplasmic ribosome or by transfer of a mitochondrial deletion approximately in the center of the coding sequence, gene, rather than by transfer of a chloroplast gene to the creating two overlapping open reading frames with homology nucleus. A mitochondrial origin seems unlikely, since no gene to the two halves of the tobacco gene (635, 746). Transcripts encoding the equivalent of L21 has so far been identified in a for both reading frames could be detected in vivo by S1 mitochondrial genome of any plant (371). mapping in spinach (635). However, no radioactive peptides Protein L22 binds to 23S rRNA early in assembly of the 50S corresponding to these transcripts were seen on two-dimen- particle in E. coli and is one of only five proteins both necessary sional electrophoresis of the products of a coupled transcrip- and sufficient to formation of the core precursor particle RI* tion-translation system. Furthermore, when chloroplast ribo- (459). Equivalent proteins have been identified in archaebac- somal proteins of a size close to those expected for the teria and cytoplasmic ribosomes (381, 708). The rp122 gene is chloroplast rpl23 gene products, either singly or spliced, were found in chloroplast genomes of all land plants so far exam- subjected to N-terminal sequencing, none of the sequences ined, with the exception of two unrelated groups of angio- obtained corresponded to the predicted sequence of the split sperms, the legumes and the parasitic plant Epifagus virginiana rp123 gene. Bubunenko et al. (70) have recently reported that (193, 485) (Table 3). A nuclear gene encoding L22 has been chloroplast ribosomes of spinach contain no protein that cloned from pea (193). In this gene, the exon encoding the cross-reacts with the product of the functional chloroplast rpl23 putative N-terminal transit peptide is separated by an intron gene of maize but do contain a protein with strong homology from the conserved structural gene. Gantt et al. (193) specu- to the L23 equivalent of eukaryotic cytoplasmic ribosomes. lated that the transit peptide sequence may have been acquired This is the first suggestion that a nuclear gene encoding a by a form of exon shuffling. The rp122 gene is also missing from cytoplasmic ribosomal protein has been substituted for a the relic of the S10 operon in the C. reinhardtii chloroplast nonfunctional chloroplast gene. genome (277) but is found in the expected location in the In Epifagus virginiana the plastid rpl23 sequence is also a plastid genomes of Euglena gracilis and the red algae Gracilaria pseudogene (714). However, Yokoi et al. (732) found that the tenuistipitata and Porphyra purpurea (86, 295, 514) and in the tobacco rpl23 gene, which does have a continuous open reading cyanelle genome (163, 423). frame, appears to be functional, since the N-terminal sequence The spinach rp122 gene encodes a protein with a central of a 13-kDa protein from the 50S ribosomal subunit exactly region homologous to all L22 proteins but has N-terminal and matches that predicted from the chloroplast rp123 gene. The C-terminal extensions with structural similarity to the E. coli rp123 genes from three monocots (rice, wheat, and maize) are L18 and L25 proteins on the basis of hydropathy profiles (651, also uninterrupted, and their derived amino acid sequences are 741). The spinach L22 protein binds to SS rRNA, protecting virtually identical to one another. In rice an rp123 gene is three nonoverlapping binding sites (76, 651). In E. coli, how- located in the inverted repeat, but an open reading frame with ever, L22 does not bind SS rRNA but L18 and L25 do. These homology to rpl23 is also present in the large single-copy observations suggest the interesting possibility that the spinach region between rbcL and petA (265). Analysis of the corre- L22 protein ("CS-L13") serves the composite functions of all sponding region in wheat (Triticum aestivum) and two closely three of these proteins. Carol et al. (76) have shown that the related plants,Aegilops squarrosa andA. crassa, has revealed an L22-like central domain of the spinach protein is required for apparent rp123 pseudogene in wheat andA. crassa but not inA. SS binding, so that this domain appears to have a function squarrosa, whose chloroplast genome seems to have sustained lacking in the E. coli protein. However, both the spinach and E. a deletion in this region as a result of illegitimate recombina- coli L22 proteins bind erythromycin. The monocots rice and tion between short direct repeats (45, 46, 469). Wheat has a maize have L22 proteins with similar 29-residue N-terminal length polymorphism just downstream of this gene compared extensions, which, however, have little homology to the spinach with A. crassa, also apparently the result of an illegitimate extension. N-terminal extensions are lacking in the L22 pro- recombination event between relics of short repeats. Se- teins of tobacco, Marchantia, Euglena, and Gracilaria species; quences with strong homology to the chloroplast rpl23 genes the tobacco protein has a C terminus that is longer than that of and pseudogenes have also been detected in mitochondrial Marchantia polymorpha but considerably shorter than that of DNA of rice and maize (45). spinach. The rpl23 gene is missing from the corresponding operon of The position of L23 in the E. coli ribosome has been the cyanelle genome (32, 598). In Euglena gracilis, the rp123 controversial, with cross-linking and immunoelectron micros- gene is in the expected position at the start of the "S10" operon copy studies giving conflicting results (91, 447). Kruft et al. but is interrupted by three group III introns (243). An unin- (328) propose that it has an elongated structure, with the terrupted rp123 gene has been sequenced from the chloroplast N-terminal domain close to L29 at the base of the 50S subunit genome of C. reinhardtii, in the expected position at the start of and the C-terminal domain on the ribosomal surface close to the "S10" operon (277). However Randolph-Anderson et al. the peptidyltransferase center. Proteins equivalent to E. coli (510) found that antibody to C. reinhardtii "L-29," a cytoplas- VOL. 58, 1994 CHLOROPLAST RIBOSOMES AND PROTEIN SYNTHESIS 729 mically synthesized protein, cross-reacts with E. coli L23 and tive (103). A nuclear gene encoding a protein with sequence with spinach ribosomal protein "L28." This antibody showed similarity to E. coli protein L28 has been isolated from tobacco no cross-reaction with Anabaena "L24," a protein which (731). The "L-31" protein of C. reinhardtii chloroplast ribo- comigrates with E. coli L23 on two-dimensional gels. somes was observed to comigrate with E. coli L28, but immu- L24 is an early-assembly rRNA-binding protein in bacteria, nological cross-reactivity was not tested (510). which, together with L3, is essential for initiation of assembly E. coli protein L29 also can be cross-linked to the 5' domain of the 50S subunit but seems not to be essential either for late of the 23S molecule (57) but associates with the ribosomal stages of ribosome assembly or for translation in vivo. An E. particle early in the assembly process (459). L29 appears in the coli mutant lacking L24 can grow, albeit very slowly (257, 461). same ribosomal neighborhood as proteins L2, L4, L15, and A reading frame specifying L24 has been found in the chloro- L34 (676). A gene encoding an equivalent chloroplast protein plast genome of P. purpurea, in the position expected based on has so far been found only in Porphyra purpurea (514). similarity to the E. coli spc operon (514), but this protein Protein L30 of E. coli assembles late and can be eliminated appears to be nucleus encoded in land plants, and cDNAs by mutation (103, 459). An equivalent protein has been encoding it have been sequenced from pea (192), spinach (75, identified in archaebacteria (708) but so far not in chloroplasts. 339), and tobacco (153). The plant sequences have transit L31 is a late-assembly protein in E. coli (358, 459) and is known peptides of about 70 amino acids, as well as highly conserved so far only from the Porphyra chloroplast genome. C-terminal extensions. E. coli L32 also associates with the ribosome late in the L25 is a 5S-binding protein in E. coli (57). No exact homolog assembly process (459). Genes encoding L32 have been found has been identified in chloroplast ribosomes. However, in in the chloroplast genomes of several plants and algae (Table spinach two chloroplast ribosomal proteins, L22 and "CS-12," 3). However, this gene is missing from the Epifagus chloroplast have been shown to bind SS rRNA and may thus together serve genome (714). Deduced amino acid similarity to the E. coli the same function as E. coli L5, L18, and L25 (76, 651) (see gene is low (Table 3), but hydropathy plots suggest that the above). The 5S-binding domain of spinach "CS-12" shows plant and bacterial proteins are similar in conformation (733). structural similarity to that of L25. The N-terminal portions of the chloroplast L32 proteins are The protein formerly designated as L26 of E. coli is now highly conserved in amino acid sequence, whereas the C- identified as S20 (706). terminal ends are variable in sequence and in length. L27 is a conserved protein that maps by immunoelectron The E. coli L33 protein can be cross-linked to 23S rRNA at microscopy to the base of the central protuberance of the 50S positions 2422 to 2424 (481) and to proteins Li and L27 (676). subunit of the E. coli ribosome and appears to be associated Mutants of E. coli lacking L33 are viable but cold sensitive with the peptidyltransferase center (57, 744). Plastid genes (103). The rp133 gene is chloroplast encoded in land plants and encoding this protein have been sequenced from chromophyte Porphyra purpurea and has also been sequenced from the and rhodophyte algae (185, 517), but the gene appears to be cyanelle genome (Table 3) but is missing from the Euglena nucleus encoded in green algae and in land plants. Two cDNAs chloroplast genome (243). encoding a protein homologous to E. coli L27 have been Protein L34 has been characterized in a number of eubac- sequenced from tobacco (154) and found to have differing terial species but has not yet been identified in chloroplasts 3'-flanking sequences, suggesting that the tobacco nuclear except for those in Cyanophora and Porphyra purpurea. In the genome encodes more than one L27 gene. The identity of E. coli ribosome, it is found in a neighborhood with proteins these cDNAs was confirmed by comparing the predicted amino L2, IA, L15, and L29 (676). acid sequences with that determined for the purified L27 Protein L35, formerly designated ribosomal protein A in E. protein (154). N-terminal amino acid sequencing of the cyto- coli (675), is encoded in the nuclear genome of spinach (577) plasmically synthesized ribosomal protein designated "L-18" in but in the chloroplast genome of P. purpurea (514) and in the C. reinhardtii (547) indicates that this protein is the E. coli L27 cyanelle genome of Cyanophora paradocxa (69). homolog (363), but the gene has not yet been cloned. Schmidt L36, the product of the E. coli gene formerly designated secX et al. (545, 546) found that C. reinhardtii "L-18" is synthesized (675), is chloroplast encoded in all plants and algae so far as an 18.5-kDa precursor that undergoes a two-step processing examined (Table 3). The high degree of conservation of the reaction. Conversion of the 17-kDa intermediate identified in amino acid sequence of the chloroplast L36 proteins compared pulse-labeling experiments to the mature 15.5-kDa form re- with E. coli (Table 3) suggests that this small protein may have quires chloroplast protein synthesis. Liu et al. (363) showed an important but as yet unknown role in the ribosome. that the 17-kDa intermediate specifically associates with a ribosomal complex that migrates with the ribosomal large Chloroplast Ribosomal Proteins with No Obvious subunit before being processed to the mature protein. This Homology to Those of E. coli suggests that the second processing step may be required for maturation of the 50S ribosomal subunit. Antibody to C. Zhou and Mache (741) reported that spinach chloroplasts reinhardtii "L-18" cross-reacts with E. coli L27, with spinach contain relatively large amounts of a unique ribosomal protein, "L22" (terminology of Mache et al. [378]), and with an "CS-S5." The deduced amino acid sequence of a full-length Anabaena protein ("L23" [510]). Elhag and Bourque (154) cDNA clone for the nuclear gene encoding this ribosomal show the alignments of the tobacco L27 sequence with the protein shows no sequence similarity to any bacterial ribo- partial L27 sequences of C. reinhardtii (547) and spinach (607), somal protein (28, 741). Working independently, Johnson et al. and with that of the yeast mitochondrial ribosomal protein (290) characterized a 26-kDa protein from spinach chloroplast MRP7 (167). Elhag and Bourque (154) note that this is the first ribosomes ("PSrp-1"), which appears to be identical to "CS- example of a chloroplast ribosomal protein for which the SS." The DNA sequences determined by the two groups differ sequence of a presumably homologous mitochondrial ribo- in three nucleotides, one of which creates a frame shift that somal protein is known. changes the predicted C-terminal sequence. Direct sequencing L28, which cross-links to the 5' domain of the 23S rRNA, is of protease-generated internal peptides supports the 236- added to the E. coli ribosome relatively late in assembly (57, amino-acid sequence published by Johnson et al. (290). The 459), and mutants lacking L28 are viable although cold sensi- precursor form of this protein contains 302 amino acids. 730 HARRIS ET AL. MICROBIOL. REV.

Lagrange et al. (339) proposed to designate this ribosomal the ribosome. Similar analyses should be possible with other protein S22, since S21 is the highest-numbered protein of the ribosomal proteins. E. coli small subunit. Schmidt et al. (541) have proposed alternatively that the numbers 22 to 29 be skipped and that this ASSEMBLY OF CHLOROPLAST RIBOSOMES protein be named S30 instead. Schmidt et al. (541) and Wada et al. (674) have indepen- Subramanian (606) points out that the ribosomal proteins dently identified another novel protein in preparations of encoded in land plant chloroplast genomes share the following spinach chloroplast ribosomes. Schmidt et al. described a basic properties: (i) all are important proteins in early steps in protein of about 7.5 kDa and gave it the designation S31. A ribosome assembly as judged from comparison with assembly sequence of 43 of the estimated 60 amino acids constituting maps of the E. coli 30S and 50S subunits (see, e.g., reference this protein showed no homology to any known E. coli 707); (ii) their loss is likely to be lethal, since no E. coli mutants ribosomal protein or to any other sequence available in public lacking any of these proteins, with the exception of L33, have databases, nor did it correspond to the derived amino acid been isolated; and (iii) all are basic or highly basic ribosomal sequence of any coding region in a published chloroplast DNA proteins, even though chloroplast ribosomes contain a much sequence. However, the sequenced region was shown to have larger number of acidic ribosomal proteins than E. coli ribo- 42% identity to the unpublished sequence of a small basic somes do. protein isolated from the bacterium Thermus thermophilus. The limited data available on chloroplast ribosome assembly Wada et al. (674) described a 5-kDa protein, SCS23, with no have been summarized for land plants by Mache (377). The apparent homology or immunological cross-reactivity to any E. main observations on land plants and C. reinhardtii are as coli ribosomal protein. The N-terminal sequence of this pro- follows. (i) Seven chloroplast ribosomal proteins, four of which tein is similar to that published by Schmidt et al. (541). are made in the chloroplast, bind to chloroplast or E. coli 16S Two cDNAs isolated from pea encode ribosomal proteins of rRNA, in agreement with the seven E. coli ribosomal proteins moderate size with no recognizable similarity to any ribosomal known to bind to 16S rRNA (531). (ii) Two 5S rRNA-binding protein of E. coli (192). The PsCL18 gene encodes a protein of proteins have been detected in spinach (L22 and "CS-12" 145 amino acids, including a transit sequence of approximately [651]), in contrast to three in E. coli (L5, L18, L25). However, 50 amino acids, and the PsCL25 gene specifies a protein of 104 one of these chloroplast proteins, encoded by the rp122 gene, amino acids, of which about 30 amino acids constitute a transit has a central region of homology to other L22 proteins flanked sequence. Typical of ribosomal proteins, the deduced amino by long N- and C-terminal extensions (76). (iii) In C. rein- acid sequences of both PsCL18 and PsCL25 have a high hardtii, the second step of processing of "L-18," a homolog of content of lysine and arginine residues, and a consequent high E. coli L27, occurs during ribosome assembly and may be net positive charge, but differ in the distribution of these required for maturation of the 50S ribosome subunit (363). (iv) charged amino acids. PsCL18 has a highly charged, highly basic The nucleus-encoded ribosomal protein "L-29" of C. rein- carboxyl end, whereas the carboxyl terminal of PsCL25 con- hardtii is required for assembly of chloroplast-encoded ribo- tains mostly uncharged amino acids with four aspartic acid somal protein "L-13" (see below and reference 446). residues constituting the only charged species. No E. coli Mutants with defects in chloroplast ribosome assembly have ribosomal protein has a carboxyl terminus resembling either of been identified in C reinhardtii and map to seven nuclear and these nucleus-encoded chloroplast ribosomal proteins from two chloroplast loci (see reference 247 for a summary). Two pea. A protein similar to PsCL18 has been isolated from phenotypic classes are seen, one in which small subunits are spinach and designated L40 (75, 339). This protein appears to deficient but large subunits accumulate and one in which both be encoded by a single-copy nuclear gene and to contain 142 subunits are deficient. No mutant specifically deficient in large amino acids with 54% sequence identity to pea CsL18. This is subunits has been identified. Analysis of double-mutant com- a slightly lower sequence identity than is seen between other binations of five nonallelic nuclear mutations led to the nucleus-encoded ribosomal proteins of higher plants, e.g., S17, proposal that mutants deficient in both subunits were blocked L12, and L15 (Table 3). in steps common to the assembly of the two subunits, while the mutants that accumulated large subunits were blocked only in Comparative Analysis of Ribosomal Proteins the assembly of small subunits (248). One of the mutants deficient in both subunits, ac-20, has subsequently been shown Sequence comparisons across phylogenetic lines can reveal to be defective in its ability to splice an intron present in the essential structural features of both RNAs and proteins. This precursor of 23S rRNA (259). Since expression of the mutant technique has been beautifully exploited in establishing con- defect apparently occurs after processing of the primary rRNA served loops and helices in 16S and 23S rRNAs (see, e.g., transcript, these observations suggest that inability to process reference 236) but has been less well developed to date in pre-23S rRNA properly results in a deficiency of large sub- analysis of ribosomal proteins. Golden et al. (213) have units, which in turn prevents small subunit assembly. recently reported the three-dimensional structure of ribosomal Two Chlamydomonas allelic nuclear mutations, cr-6 and protein S17 from B. stearothermophilus, based on nuclear cr-7, cause production of ribosomal large subunits that sedi- magnetic resonance spectroscopy, and Hoffman et al. (266) ment abnormally on sucrose gradients, assemble into mono- have solved the crystal structure for protein L9 from this mers less efficiently than those from wild-type cells, and show bacterium. The comparative analysis presented in both these reduced capacity for protein synthesis in vivo (446). Large papers includes the sequences for the homologous proteins subunits of chloroplast ribosomes from these two mutants lack from pea and Arabidopsis thaliana, as well as the Cyanophora two proteins, one of which ("L-29") is made in the cytoplasm S17 and Synechocystis L9 sequences. Conserved structural and the other ("L-13") is made in the chloroplast. The primary residues and proposed rRNA-binding sites can be identified in defect appears to be an inability to make "L-29," which both proteins. Conservation of the length of an al-helix in the prevents assembly of "L-13" into the 50S subunit. Immunolog- L9 protein, for example, suggests that this helix has a structural ically, "L-29" is related to E. coli L23 and to a lesser extent to role, whereas variability in the central region of the protein L7/L12, while "L-13" is related to E. coli L5 (see above and sequence is consistent with its occupying an exposed position in reference 510). Assembly of L5 into the E. coli 50S subunit VOL. 58, 1994 CHLOROPLAST RIBOSOMES AND PROTEIN SYNTHESIS 731 does not depend directly on the presence of either L7/L12 or and that the 5S rRNA is rapidly cleaved from the initial L23 (707). precursor (10, 601). Possible promoter sequences have been Chloroplast and nuclear mutations causing complete or found between the 4.5S and 5S rRNA genes in some plants, nearly complete loss of chloroplast ribosomes from white suggesting that separate 5S transcription might occur, but tissues and seedlings have also been identified in land plants these sequences are not present in many plants and do not (39) and have been very useful in probing the function of the seem to be active in vitro (10, 146). Likewise, the tRNA'g chloroplast protein-synthesizing system. Barkan (17) has re- following the 5S gene in land plants is thought to be cotrans- cently described transposon-induced nuclear mutations in cribed with the rRNAs (118). However, the tRNAVal upstream maize that impair chloroplast protein synthesis. Seedlings of of the 16S gene in many plants lies distal to the identified these mutants are paler green than those of the wild type and transcription start sites for the rRNA operon and thus is not are photosynthetically inactive, although they do accumulate part of the primary transcript. nucleus-encoded proteins of the light-harvesting complex. One Possible processing sites of the primary rRNA transcript mutant appears to be blocked specifically in processing of 16S have been identified by Si and reverse transcriptase mapping rRNA. (118, 601; see also reference 114). Experiments so far suggest Complete assembly of ribosomal subunits has been detected that cleavage at the various sites does not occur in a precise in isolated spinach chloroplasts, implying either that a pool of order. The 5'- and3'-terminal precursor sequences of the 16S unassembled nucleus-encoded ribosomal proteins exists in and 23S RNAs can form double-stranded stem structures plastids or that ribosomal proteins can be released from similar to those of the E. coli rRNA genes, leading to the preexisting ribosomes and reutilized (121). Certain proteins, suggestion that these molecules may be processed by an RNase e.g., the CS-S5 protein of spinach, can be found in high III-like endonucleolytic cleavage (114). Vera et al. (669) have concentrations in the chloroplast stroma (741). When rye found that tobacco chloroplast ribosomes contain a minor plants are grown at high temperature (32°C), plastid ribosome fraction of 16S rRNA molecules in which a 30-nt leader formation is severely impaired (168, 169). Pools of a few sequence containing the putative RNase III site is still present, unassembled plastid ribosomal proteins were detected when suggesting that the final maturation of the 16S rRNA may soluble extracts from leaves deficient in 70S ribosomes were actually take place within the ribosome. examined with antibodies raised against purified 50S and 30S Processing of the tRNAs of the spacer between the 16S and subunits. These antibodies were shown to react with about 17 23S genes requires the RNase P (679), but other of the 33 polypeptides of the 50S subunit and 10 of the 25 processing reactions specific to plant chloroplast rRNAs have proteins of the 30S subunit. Feierabend and Berberich (168) not been well characterized to date. Additional processing of believe that these observations confirm the absence of plastid the mature 23S rRNA of land plants may occur, leading to ribosomes following bleaching and that the unassembled pro- hidden breaks at specific stem-loop sites. Thus 23S rRNA teins detected by the antibodies are probably of cytoplasmic isolated under denaturing conditions typically appears as sev- origin. eral short species rather than a single intact molecule. Delp and Kossel (114) suggest that this fragmentation is real, not an SYNTHESIS OF THE COMPONENTS OF artifact of preparation, and that it may be necessary for some CHLOROPLAST RIBOSOMES structural or functional requirement of the chloroplast ribo- some. Biogenesis of chloroplast ribosomes requires expression of There are conflicting data regarding whether chloroplast both nuclear and chloroplast genes encoding different ribo- rRNA genes are transcribed by a different RNA polymerase somal proteins, as well as chloroplast genes encoding the from that used to produce mRNAs (229, 283, 320, 349, 642). component rRNAs. The mechanisms by which the appropriate Chloroplast genomes typically contain genes homologous to stoichiometry of these components is achieved from genes the rpoA, rpoB, and rpoC genes, which encode RNA poly- present in vastly different copy numbers remain poorly under- merase subunits of bacteria, but appear to lack a gene for rpoD, stood (229, 377, 379). In general, nuclear gene expression in which encodes the principal cr factor of the bacterial RNA plants tends to be controlled at the level of transcription and to polymerase (24, 171, 614, 653). However, immunological stud- be subject to light regulation, whereas chloroplast gene expres- ies suggest that the chloroplast RNA polymerase complexes do sion is largely regulated posttranscriptionally. Pool sizes of contain a-like factors (642, 653). Expression of the chloroplast- some components may also be controlled by proteolysis. encoded RNA polymerase genes in Euglena chloroplasts re- Furthermore, chloroplast genes encoding ribosomal compo- sulted in activity of a soluble RNA polymerase fraction capable nents appear to be regulated differently from those encoding of transcribing tRNA and mRNA genes, whereas transcription proteins of the photosynthetic apparatus (207, 253, 365). In of rRNA genes required a membrane-bound fraction (226, this section we will discuss transcription and splicing of rRNAs, 244, 361). However, soluble RNA polymerase from spinach transcription and translation of genes encoding ribosomal chloroplasts appears to be able to transcribe both rRNA and proteins, and posttranscriptional and translational regulation protein-coding genes in vitro (55). Fractionation of RNA of chloroplast gene expression. polymerase activities from spinach suggests that a 110-kDa component may represent a core enzyme active as a single Transcription of rRNA Genes polypeptide chain, which shows no immunological similarity to E. coli RNA polymerase subunits and is thus probably not the As in bacteria, chloroplast rRNA genes are thought to be product of the chloroplast rpoB gene (349). Further evidence transcribed as large precursor molecules that subsequently for a second, presumably nucleus-encoded RNA polymerase undergo several processing steps to generate the mature activity in chloroplasts comes from the observation that the rRNAs (114). Relatively little is known, however, about the plastid genome of the parasitic plant Epifagus virginiana lacks specific enzymes and cleavage steps that are involved. Al- the rpo genes but is nevertheless transcribed (115, 435, 713, though 5S rRNA sequences are not detected in the primary 714). Also, some plastid genes in heat-bleached leaves of rye transcript, S1 and primer extension experiments suggest that and barley plants and the albostiians mutant of barley are the 5S gene is indeed cotranscribed with the 16S and 23S genes transcribed, although these leaves lack functional chloroplast 732 HARRIS ET AL. MICROBIOL. REV.

ribosomes and are thus unable to translate the mRNAs for the nuclear genes encoding chloroplast ribosomal proteins are plastid-encoded rpo genes (164, 262, 263). subject to light regulation. Pea seedlings grown in bright light In land plant chloroplasts, promoter sequences preceding in the presence of the inhibitor norflurazon, which blocks the transcription start sites of rRNA operons do not differ carotenoid synthesis, showed greatly decreased levels of significantly from the -10 and -35 consensus sequences of mRNA for nucleus-encoded ribosomal proteins compared plastid protein-coding genes and are typically 50 to 200 bp with seedlings grown with or without norflurazon in the dark. upstream of the 16S rRNA genes (229). The resemblance to Levels of mRNAs for other chloroplast components were promoter sequences of protein-coding genes implies that a similarly diminished, but mRNAs for cytoplasmic ribosomal single-core RNA polymerase might be able to transcribe all proteins, histones, and other nonphotosynthetic proteins were classes of RNAs, a notion that is also supported by the not affected. demonstration that a chimeric gene consisting of the 16S promoter fused to the bacterial aadA gene encoding spectino- Transcription of Chloroplast Genes Encoding mycin and streptomycin resistance is expressed in chloroplast Ribosomal Proteins transformants of tobacco (621). This construct was edited by site-directed mutagenesis to eliminate upstream AUGs in the Nuclear and plastid genes which cooperate in controlling mRNA, and a synthetic leader sequence containing a ribo- chloroplast biogenesis and function appear to be regulated by some-binding site was attached. The aadA gene was followed very different mechanisms, although their gene products often by the 3' region of the chloroplast psbA gene. However, occur in equal stoichiometry within the multimeric thylakoid deletion analysis with chloroplast transformants in which pu- complexes or chloroplast ribosomes. This may reflect the way tative promoter regions were fused to a reporter gene has led in which plant cells cope with large differences in ploidy levels to the identification of two classes of chloroplast promoters in between nuclear genes (present in single copies or small gene C. reinhardtii (310). Promoters of the first class, such as atpB, families) and chloroplast genes (present in hundreds or thou- lack a conserved -35 sequence, and deletion of this region has sands of copies per cell). Nuclear genes encoding chloroplast no effect on relative rates of transcription or the transcription polypeptides are regulated largely at the transcriptional level in initiation site. The second class of promoters includes the 16S response to environmental and developmental signals (for rRNA gene and has a conserved and essential -35 sequence. reviews, see references 209, 329, and 443). In contrast, most A 14-bp sequence that is recognized by polypeptides of 33 and plastid genes appear to be transcribed at all times during 35 kDa has also been identified upstream of the 16S rRNA plastid development, and posttranscriptional regulatory mech- initiation start site in spinach (13). This sequence is not found anisms are thought to play major roles in modulating their upstream of chloroplast genes encoding mRNAs or tRNAs expression (see below). and thus may have a role in differential regulation of rRNA All 21 of the ribosomal protein genes in the rice, tobacco, and protein-coding genes during chloroplast development. maize, and Marchantia chloroplast genomes have been dem- Expression of rRNA genes appears to depend on both light onstrated to be transcribed (540), but not all have been shown and developmental stage in plant seedlings (309, 442), but unequivocally to be translated into the corresponding polypep- steady-state levels of rRNA seem to be controlled more by the tides. N-terminal sequencing indicates that ribosomal proteins rate of breakdown than by transcriptional regulation (114). S12, S16, S19, L2, L20, L32, L33, and L36 of spinach chloro- Bendich (21) has suggested that rRNA transcription is regu- plasts do indeed appear to be products of the corresponding lated primarily by gene dosage. However, in cells of C. chloroplast genes (540). Most of the clusters of chloroplast- reinhardtii grown in the presence of 5-fluorodeoxyuridine, the encoded ribosomal protein genes that show striking homology reduction in chloroplast DNA copies was mirrored by a to bacterial ribosomal protein operons (631) (Fig. 5) are reduction in accumulation of chloroplast rRNA (208, 272). probably functional transcriptional units (540). Multiple tran- There is indirect evidence that conserved stem-loop structures scripts are typically found for individual ribosomal protein and short open reading frames found between the promoter genes in these clusters, and these may arise from processing of and the start of the 16S coding sequence could be involved in larger polycistronic transcripts (470, 605). In C. reinhardtii, no regulation of rRNA operon expression in spinach chloroplasts single large mRNA has been detected for the ribosomal (338). protein gene cluster that begins with the rp123 gene, but, Bisanz-Seyer et al. (27) observed the accumulation of 16S rather, a series of transcripts of different lengths have been rRNA and mRNAs for several chloroplast ribosomal proteins observed, including some probably monocistronic transcripts during early development of spinach. The 16S rRNA, already and others corresponding to two or more genes (362; see also present in dry , began to increase at the time of reference 253). germination 5 days after planting and continued to accumulate Christopher and Hallick (87, 88) published a detailed char- thereafter. Most ribosomal protein mRNAs appeared at the acterization of the organization and transcription of the large beginning of germination (5 days), but the rpsl9 and rp123 ribosomal protein operon in Euglena gracilis. The primary mRNAs appeared 2 days earlier. These two genes belong to a transcript of this operon includes 11 genes encoding ribosomal large chloroplast ribosomal protein operon including parts of proteins, a tRNA gene, and an open reading frame encoding a the S10, spc, and a operons of E. coli. Interestingly, mRNAs highly basic protein of unknown function (88, 243). An 8.3-kb for several other ribosomal protein genes in this operon, mRNA from which all introns have been removed by splicing including rps3 and rp116, did not begin to accumulate until is then processed stepwise into transcripts containing one or germination. One explanation of these results is that the first more genes. three genes in this operon, rp123-rp12-rpsl9, are transcribed Tonkyn and Gruissem (648) examined the relative expres- early and the whole operon is transcribed later. This hypothesis sion levels of the intact S10 operon from spinach and the predicts that rp12 transcripts should also be detected early, but partial S10 operon that begins in the opposite inverted repeat this was not examined with appropriate probes. Alternatively, but ends in an rpsl9' pseudogene as discussed above. Because the entire operon may be transcribed early, but transcripts the upstream regulatory regions of these two operons are distal to rpsl9 are initially degraded. included in the inverted repeat in spinach and are therefore Gantt et al. (194) have presented additional evidence that identical, Tonkyn and Gruissem predicted that these operons VOL. 58, 1994 CHLOROPLAST RIBOSOMES AND PROTEIN SYNTHESIS 733 might be expressed at the same level and that a nonfunctional than as transcript terminators in land plants, there is some product of the rpsl9' gene might accumulate. In fact, they evidence that they serve as terminators for chloroplast tran- found that the rpsl9' transcript was present at very low levels scripts in C. reinhardtii (30). Experiments are currently under if at all and that the rp12 mRNA that is translated appears to be way in several laboratories to demonstrate the functional role transcribed from the gene copy located in the complete of individual binding proteins in mRNA stability and transla- operon. However, transcription of the intact operon can be tion as well as to decipher the signal transduction pathway initiated from several different promoters, suggesting that it leading to the expression of these proteins. For example, may be subject to developmental regulation. Nickelsen and Link (456) have described a 54-kDa protein A number of genes encoding ribosomal proteins are part of from mustard chloroplasts that binds to a conserved sequence mixed clusters that also contain genes encoding components of in the 3'-flanking regions of the tmK and rpsl6 genes and the photosynthetic apparatus. Stahl et al. (587) demonstrated appears to have endonucleolytic activity that may be involved that such a mixed chloroplast gene cluster in maize containing in RNA3'-end formation and mRNA stability. genes encoding four subunits of the ATP synthase (atpI, atpH, Development of reliable protocols for chloroplast transfor- atpF, and atpA) and the gene encoding ribosomal proteinS2 mation in C. reinhardtii (48) and tobacco (390), coupled with (rps2) produces a total of 12 transcripts, including a major the ability to express foreign reporter sequences in the chlo- species of 6,200 nt containing mRNAs of all five genes. They roplast, has allowed the functional dissection of the 5' and 3' suggest that this plastid gene cluster is "functionally organized untranslated leaders for the first time (84, 214, 535, 591, 621). as an operon with additional regulatory features to allow for Detailed consideration of the genetic basis for translational increased accumulation of mRNAs for the thylakoid compo- regulation in chloroplasts and mitochondria and a model nents." depicting the role of a multiprotein translation complex bound The analogous operon in Euglena gracilis, containing rps2, to the 5' untranslated leaders of organelle mRNAs in modu- atpI, atpH, atpF, atpA, and rpsl8, was analyzed by Drager and lating the translational regulation are presented in a recent Hallick (134). Of these genes, all but atpH contain one or more review (207). introns, comprising in aggregate nine introns of the group III class unique to the genus Euglena and its colorless relative Membrane Binding of Chloroplast Ribosomes Astasia (87, 565), seven introns with group II structure, and one intron that matched neither category. Drager and Hallick Thylakoid-bound polysomes have been characterized in (134) found that all 17 introns are removed to yield a 5.5-kb chloroplasts with respect to their physical status and physio- mRNA spanning all six genes, from which monocistronic logical function (see references 40, 41, and 286 for reviews). transcripts are then generated, presumably by endonucleolytic High-salt washes of isolated thykakoids remove 30 to 45% of cleavage. The unique 434-nt intron in the rpsl8 gene is a the membrane-bound RNA while addition of puromycin re- complex twintron, consisting of four group III introns which leases up to 80% of the bound RNA. By analogy with the are removed in four sequential splicing reactions, some of rough endoplasmic reticulum, these results suggest that be- which can use multiple splice sites (135). tween one-third and one-half of the polysomes found on Chen et al. (83) found that the psaA, psaB, and rpsl4 genes thylakoids are attached electrostatically to the membranes and in rice are organized into a single transcriptional unit. A 5.2-kb the rest are held by both electrostatic forces and nascent transcript hybridizing to probes for all three genes was ob- polypeptide chains. The electrostatic binding of chloroplast served in leaf tissue. Ribosomal protein L32 of the tobacco polysomes predicts the presence of a ribosome receptor similar chloroplast has been shown to be encoded by the gene to the ribophorin-containing receptor found on the rough formerly identified as open reading frame ORF55 (733), endoplasmic reticulum (503) but missing from the bacterial located in the small single-copy region. A primary transcript of cytoplasmic membrane. Other components of the system for 1,550 nt contains no other open reading frames and overlaps synthesizing eukaryotic secretory proteins, such as a signal the ndhF gene on the opposite strand (668). Vera et al. (668) recognition particle and a docking protein, have not been demonstrated that the rp132 promoter is located within the demonstrated in chloroplasts. The fraction of membrane- ndhF coding region, the first instance so far of an internal bound polysomes observed can be markedly enhanced by divergent promoter in the chloroplast genome. pretreating the cells with antibiotics such as chloramphenicol and erythromycin that inhibit transpeptidation. Posttranscriptional Regulatory Mechanisms Freimann and Hachtel (180) examined the distribution of Affecting Chloroplast mRNAs mRNAs on free and membrane-bound chloroplast polysomes of broad bean (Vicia faba). They used the criteria of release of Recent reviews by Rochaix (520), Gruissem and Schuster the associated mRNA by high salt alone or high salt plus (228), and Gruissem and Tonkyn (229) provide excellent puromycin, together with gene-specific probes, to distinguish summaries of the current literature dealing with the process- mRNAs electrostatically bound to thylakoids from those en- ing, stability, and translational control of chloroplast mRNAs gaged in cotranslational protein synthesis. Three classes of in general, although relatively little of this literature is specific mRNA were recognized. (i) The rbcL mRNA encoding the to genes encoding ribosomal proteins. Two generalizations large subunit of Rubisco was the only mRNA associated solely emerge from these reviews. First, nuclear gene products con- with stromal polysomes. However, other authors have reported trol expression of chloroplast-encoded mRNAs. On the basis that rbcL mRNA is also found associated with thylakoid of analysis of nuclear nonphotosynthetic mutants of C. rein- membranes (252). (ii) Thylakoid polysomes containing hardtii, several different gene products may be required for mRNAs for six genes encoding integral membrane proteins expression of a given chloroplast mRNA (520). Second, spe- appeared to synthesize their products in a cotranslational cific proteins bind to inverted repeat regions present in the 5' fashion. These mRNAs were released only by high salt plus and 3' untranslated leaders of chloroplast mRNAs that are puromycin. (iii) Thylakoid polypeptides encoded by seven capable of forming thermodynamically stable stem-loop struc- other genes were assumed to be incorporated posttranslation- tures (105, 229). Although the 3' untranslated leaders appear ally because their mRNAs were found on stromal polysomes or to function in stabilization and processing of mRNAs rather polysomes bound electrostatically to the thylakoid membranes. 734 HARRIS ET AL. MICROBIOL. REV.

Other supporting evidence exists that such chloroplast-synthe- components of the plastid gene expression system (rRNAs, sized proteins as Dl, the reaction center polypeptide encoded tRNAs, and ribosomal proteins). Functional photosynthesis by the psbA gene, and the alpha and beta subunits of the CF1 genes and genes of the NADH dehydrogenase complex are portion of the ATP synthase are made, at least in part, on absent, although several photosynthetic pseudogenes have thylakoid-bound polysomes (see reference 286 for a summary). been found. The Epifagus plastid genome contains only 17 Membrane binding of polysomes may play an additional role tRNAs, suggesting that tRNAs must be imported if this plastid in translational regulation of chloroplast gene expression. In C. protein-synthesizing system is to function. Genes specifying the reinhardtii, the distribution of chloroplast mRNAs varied four RNA polymerase subunits encoded in the chloroplast between the thylakoid and soluble fractions in cells growing genomes of green plants are also absent. Although no exper- synchronously on a light-dark cycle (41). Thus, a striking iments with Epifagus virginiana demonstrating synthesis of a increase in the fraction of membrane-bound polysomes was specific plastid-encoded protein have been reported, there are observed for both rbcL and psbA mRNAs in the light period. several lines of indirect evidence suggesting that the plastid Thylakoid binding may occur in the light phase because protein-synthesizing system is functional. Wolfe et al. (714) translation is initiated. In contrast, Klein et al. (308) found that reported transcription of all eight rRNA and protein-encoding the psaA, psaB, and psbA transcripts are primarily membrane genes so far examined and cited the following three evolution- associated in dark-grown barley plants. The protein products ary arguments in favor of function. (i) Plastid gene deletions in of these genes are made in the dark but are unstable in the Epifagus virginiana are not random but are skewed toward absence of chlorophyll (441). Jagendorf and Michaels (286) photosynthetic genes. Although only 5% of photosynthetic correctly point out that the possible role of the thylakoid sequences have been retained with respect to tobacco, 80% of membrane itself in translational regulation requires further the ribosomal protein sequences are present. (ii) Large open investigation. reading frames are retained in the Epifagus plastid genome. If these genes were nonfunctional, mutations, truncations, and internal deletions would have been expected to occur, as is true HOW ESSENTUIL IS CHLOROPLAST of pseudogenes in the Epifagus plastid genome. (iii) The PROTEIN SYNTHESIS? genera Conopholis and Epifagus share the loss of the photo- Chloroplast protein synthesis has long been known to be synthetic and ndh genes, but their rRNA genes are strongly indispensable for survival of plants and algae that depend on conserved, suggesting that the evolution of these genes is CO2 as their sole carbon source, since numerous proteins constrained by because they are functional. required for photosynthesis are plastid gene products. How- Why might the protein-synthesizing systems of Epifagus ever, as we learn more about the genes encoded in the plastid virginiana and other colorless plants be essential? The argu- genomes of algae such as Cyanophora, Cryptomonas, and ment of Howe and Smith (275) that plastid protein synthesis Porphyra species, some of which specify proteins required for was retained in Epifagus species for the sole purpose of making amino acid or fatty acid biosynthesis, the likelihood is increas- the chloroplast-encoded RNA polymerase subunits required ing that chloroplast protein synthesis is also required for the for transcription of the tRNAGIU gene necessary for porphyrin production of one or more essential proteins not involved in synthesis (see, e.g., references 20 and 552) is invalidated by the photosynthesis. This viewpoint is supported by some, but not finding that the RNA polymerase subunit genes are absent all, analyses of plastid genome function in colorless plants. We from the Epifagus plastid genome (714). However, one or more begin with cases that support the hypothesis that chloroplast other proteins essential for survival might be encoded and protein synthesis is essential and then turn to evidence that translated in the Epifagus chloroplast. The best candidate is makes the converse argument. clpP, which specifies one subunit of the plastid homolog of the The colorless heterotroph Astasia longa is closely related to ATP-dependent Clp protease of E. coli. Perhaps this protease Euglena gracilis and possesses a circular 73-kb plastid genome. is involved in processing chloroplast protein precursors into an This genome is the counterpart of the larger (145-kb) Euglena active form or in protein degradation. chloroplast genome, and the genes identified include the The thesis that chloroplast protein synthesis is essential is rRNAs, tufA, and several tRNAs and ribosomal proteins also supported by work on the genus Plasmodium, the malaria (565-569). The rbcL gene encoding the large subunit of the parasite. This protozoan contains, in addition to its tiny (6-kb) enzyme Rubisco is the only photosynthetic gene so far detected linearly reiterated mitochondrial genome, a 35-kb circular in the Astasia plastid genome. This polypeptide has been DNA molecule that seems to be a residual plastid genome (see immunoprecipitated from Astasia longa, suggesting that the references 273, 484, 700, and 701 for reviews). The 35-kb circle rbcL gene is transcribed and translated and that the plastid possesses inverted repeats containing continuous rRNAs with protein-synthesizing system of this colorless flagellate must be secondary structures quite similar to those predicted for E. coli functional. Colorless, heterotrophic algae of the genus Poly- (166, 197, 198). It also encodes tRNAs, two subunits of a toma, closely related to or derived from the genus Chlamydo- eubacterial-type RNA polymerase, and at least four ribosomal monas, contain a plastid genome (ca. 200 kb) similar in size to proteins (165). the C. reinhardtii chloroplast genome (574, 670). Plastid rRNA Genetic evidence suggests that chloroplast protein synthesis genes are present and expressed in species, leuko- in C. reinhardtii is essential for survival. Hanson and Bogorad plast ribosomes have been isolated, and the tufA gene has been (245) showed that cells carrying a nuclear mutation conferring identified. These results suggest that Polytoma species too have erythromycin resistance on chloroplast ribosomes underwent a a functioning plastid protein-synthesizing system (575, 576, marked reduction in chloroplast ribosome content when 670). shifted from 25 to 15°C. Ribosome loss was accompanied by Plastid genomes of the colorless plants Epifagus virginiana loss of the ability of the mutant to grow at 15°C under all (beechdrops) and Conopholis americana in the Orobran- conditions. Also, although several Chlamydomonas mutants chaceae family of -parasitic angiosperms have also been which have a reduced content of chloroplast ribosomes have examined (115, 702-704, 711, 712, 714). The 70-kb Epifagus been isolated, none is completely deficient in chloroplast plastid genome has been completely sequenced and contains protein synthesis (247-249, 446). Lastly, mutations with sym- only 42 genes (714). At least 38 of these genes encode metric deletions of the psbA gene encoded within the inverted VOL. 58, 1994 CHLOROPLAST RIBOSOMES AND PROTEIN SYNTHESIS 735 repeat are frequently isolated, but the only known deletion the tRNAGlU gene would require functioning of a nucleus- mutation affecting the rRNA gene region of the repeat re- encoded plastid RNA polymerase. Retention of small amounts moved only one set of rRNA genes (486). Many years ago, of plastid DNA in bleached Euglena mutants (254) is probably Blamire et al. (29) reported that treatment of wild-type cells not related to a general requirement for tRNAG'U in porphyrin with antibiotics blocking translation on chloroplast ribosomes synthesis, since mitochondrial heme in this flagellate is made inhibited replication of nuclear but not chloroplast DNA. via the animal-type 8-aminolevulinic synthetase pathway which Inhibition did not occur in mutant strains with chloroplast does not require tRNAGlU (268). In fact, the rRNA genes were ribosomes resistant to these antibiotics. These intriguing ex- the only ones detected in these deleted plastid genomes. periments have never been repeated. The existing data currently suggest that chloroplast protein The notion that chloroplast protein synthesis is indispens- synthesis may be essential for survival in Chlamydomonas and able is challenged by several other findings. Cuscuta reflexa Epifagus species but possibly not in other plants such as () is a colorless parasitic plant, unrelated to the tobacco, at least in tissue culture. The plastid-encoded tRNA- genus Epifagus, that contains residual thylakoids and traces of Glu gene is essential for synthesis of all porphyrins in plants and chlorophylls a and b (376). The plant also possesses very low algae examined to date, with the exception of Euglena gracilis, levels of light-stimulated CO2 fixation and Rubisco activity, so transcription of this gene is crucial to survival. However, the although the Rubisco large subunit is undetectable by immu- Epifagus results suggest that in this plant, at least, transcription nological methods (237, 376). Partial sequence analysis of the of tRNAGlU depends on a nucleus-encoded RNA polymerase. Cuscuta reflewa plastid genome (33, 237, 238) revealed that although many photosynthesis genes are intact (e.g., atpB, CONCLUSIONS atpE, rbcL, and psbA), a large deletion has removed certain protein synthesis genes (rpl2, rp123, and several tRNA genes), Analysis of chloroplast sequences has been invaluable in and the ndh genes appear to be nonfunctional. Transcription determining variable and conserved regions of the 16S and 23S analysis showed that rbcL was weakly transcribed while psbA rRNA molecules and in predicting their secondary and tertiary was transcribed strongly. Bommer et al. (33) hypothesized that structures. Similar comparisons of ribosomal protein se- the translational apparatus of Cuscuta reflexa is nonfunctional quences are just beginning but will doubtless prove important based on the absence of specific protein synthesis genes from in years to come. Specific domains conserved over a wide the plastid genome and their inability to detect Rubisco large variety of organisms are likely to be important in ribosome subunit using immunological techniques. Alternatively, the function or assembly. Sequence analysis of ribosomal proteins plastid protein-synthesizing system of Cuscuta reflexa might be in a diverse array of algae and land plants will allow further functional, with the missing tRNAs and ribosomal proteins refinements in understanding which domains are important for encoded in the nucleus. ribosome function. Although no analysis of mitochondrial In tobacco, antibacterial antibiotics such as streptomycin ribosomal proteins has been included here, these will also be a and lincomycin cause bleaching, but they do not cause death of valuable comparative tool in such research. We hope that this callus in tissue culture (389). The bleached antibiotic-sensitive review will provide a useful starting point for investigations on cells continue to divide at a reduced rate, using sucrose as the these topics. carbon source. Mutants resistant to these antibiotics are green in tissue culture. Streptomycin-resistant mutants result from ACKNOWLEDGMENTS specific base pair changes in the 16S rRNA or rpsl2 genes, while those resistant to lincomycin arise because of a specific We thank Hans Bohnert, Donald Bryant, Robin Gutell, Claude base pair alteration in the gene encoding the 23S rRNA (391, Lemieux, Xiang-Qin Liu, Michael Reith, Alap Subramanian, and of bleached antibiotic-sensitive cells Monique Turmel for sharing unpublished data. 590) (Table 2). The ability Our work described in this review was supported by NIH grant to continue to divide implies that chloroplast protein synthesis GM-19427. may be required only for the manufacture of photosynthetic and ribosomal proteins in Nicotiana species. However, tobacco REFERENCES calli containing a chloroplast mutation to streptomycin resis- 1. Akkaya, M. S., and C. A. Breitenberger. 1992. Light regulation of tance grow better in the dark on antibiotic than do sensitive protein synthesis factor EF-G in pea chloroplasts. 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