Plant Molecular Biology 6: 2 6 5 - 2 7 0 , 1986 © 1986 Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands

Sequence organization of the chloroplast ribosomal spacer of Chlamydomonas reinhardii: uninterrupted tRNAile and tRNAala genes and extensive secondary structure* M. Schneider & J. D. Rochaix Departments of Molecular Biology and Plant Biology, University of Geneva, 1211 Geneva, Switzerland Keywords: Chlamydomonas reinhardii, chloroplast ribosomal spacer, tRNAile, tRNAala gene

Summary The 1 805 bp spacer between the chloroplast ribosomal 16S and 7S RNA genes of Chlamydomonas reinhardii has been sequenced. It contains the genes of tRNA ala and tRNA ile which are both uninterrupted. The spacer includes several short direct and inverted repeats and a large palindromic structure which maps in the region where DNA rearrangements have occurred in other Chlamydomonas species.

Introduction

roplasts of Euglena gracilis (6, 18), tobacco (24), maize (8), bean (14), broad bean (15) and wheat (16). The spacers of Euglena gracilis (6, 18), tobacco (24) and maize (8) have been entirely sequenced. In higher plant chloroplasts the ribosomal spacer ranges between 1700 and 2400 bp, considerably longer than those of Euglena chloroplasts (259 bp) and of E. coli (437 bp). The large size of the spacers from higher plants is due mainly to the introns in the tRNA genes which range between 707 and 949 bp (8, 24). Since the C. reinhardii spacer has a size comparable to that of higher plants and since it is known to hybridize with 4S RNA (12), it was of interest to examine the structure of this spacer in more detail. Here we present the complete sequence of this spacer. It consists of 1 805 bp and it contains the genes for tRNA ala and tRNA ile both of which lack introns.

In several higher plants and algae the chloroplast ribosomal units are located within a large inverted repeat of the chloroplast genome (26). In C. reinhardii the two 19 kb segments of the inverted repeat are separated by two large single copy regions of nearly equal size (20). The ribosomal unit of this green alga consists, in the order of transcription, of the 16S, 7S, 3S, 23S and 5S rRNA genes (22). Unique features of this unit include the presence of an 888 bp intron in the 23S rRNA gene and of the two small 3S and 7S rRNA genes which precede the 23S rRNA gene (22). While higher plant chloroplasts contain a 4.5S rRNA gene between the 23S and 5S rRNA genes (26), this is not observed in C. reinhardii (22). The arrangement and the sequences of the chloroplast rRNA genes are remarkably related to those of prokaryotes (3, 23, 25). In E. coli the spacer between the 16S and 23S rRNA genes in the seven ribosomal operons contains either a tRNA glu gene or both tRNA ala and tRNA ile genes (11, 28). These two tRNA genes have also been found in the ribosomal spacer in Bacillus subtilis (10), in the cyanelles of Cyanophora paradoxa (9), in the chlo-

Materials and methods The recombinant plasmid HRI.14 containing most of the chloroplast ribosomal spacer of C. reinhardii has been described (22). Plasmid DNA

* Paper presented at the First International Congress of Plant Molecular Biology (Savannah, GA, 1985).

265

266 was prepared by the method of Katz et al. (7). D N A fragments were 3' or 5 '-endlabelled and sequenced by the chemical cleavage method as described (13). The D N A sequence analysis was performed on a Hewlett Packard computer, model 9845.

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Results and discussion Fig. 1. Restriction map and sequencing strategy of the chloroplast ribosomal spacer of C. reinhardii. The genes of tRNAile

The plasmid HRI.14 contains a chloroplast EcoRI-HindlII fragment that covers most of the spacer between the 16S and 7S rRNA genes. The first 67 bp of this spacer that flank the 3' end of the 16S rRNA gene were determined previously (4). Figure 1 displays the physical map of the spacer

and tRNAala, and the 3' and 5' ends of the 16S and 7S rRNA genes, respectively, are indicated. Restriction sites are indicated by • EcoRI, o AluI, • DdeI, ~ Hinfl, • KpnI, o TaqI.

RTTTTTRRCC~TTRTGGGTRTRTR~TR~GRGTTTTRGCTRTRRR~T~RRCTRRRG~TR~GTGGTTGR~TTCcCRCGTTRCCTTTT~AR .i~o

.15o

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~CTcRTTRTRRRRRTTTRTRRTR~RTRRRTR~R~TRR~TTRRTRRTTRRTTR~T~G~TRn~R~GGQcRRTRRRTRRRT~

TGTcCCCTT~CCTT~CGGGR~RRTRRRTRRRTTTGTTG~TG~RRcTGCCTCCTTCGGR~T~TTRRRRTC~TRTRTTTRTRTR~T~G .~o0

.3~o

.geo

RR~G~GTcCcCTTC~GsCR~TRRRTTTTRGTGGcRsTTG~T~G~cT~TcGs~TRRc~RGTTCc~T~GsRGTR~RT~T~TRsG~TGT t~Y.

• ~,o

.,~o

.,,o

TRRTRCTG~GRTR~RcTTTRGTTGCCCGRRGGGGTTTRCRTRCTCCGRRGGRGGGRG~RGG~RGTGGCGGTRCCR~T6~R~TGs~GT~ IR2

.--o

.,,o

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~ G~A~CTGC~TRGGCRRGRRR~TTRGGG~TTTTRRTG~RRTRRRTRRRTTTGTC~CCTTCGGGTRRRTRRRTTTTRGTGGRcGCCRGTG .$70

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.--o

.o.o

....

TRTCG~RGTRTTRRcRTcCTRTTTRRT~CTcGTRRRTTTRTTTG~TGcGCRsCRGGTTT~CRTRCTcC~RGGRGGRRGG~sGCR~TT~G .750

.780

.s~o

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.~,o

IR7

~CRC~RRRRTT~RTTBCCCG~G~RC~RCTRRTRTTTRTRTC~TR~GGGRC~T~CTT~CCCTTC~CC~CGGG~C~T~BG~C ¢ .gad .g~O

.~oo

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~TT~cCTGCcRRC~RTTTRTTTRTT~TRTTRRcR~CCT~nTRTRRnT~TT~GGCC~GTRRnCTTRGGRGCC~RTRRRTRTCcCGTRCG .Io~o

Io~o

.~oeo

~Gn~5~cRcTG~CB~ccTn~¢RRGTRRR~TTRGGG~RTRTRRRTRTCRRcTGGCGTccCC~TRCGGGTRcRTR~RT5T~cTRRRcTT

CCT~TTCGT~CG~GRR~R~T~R~GTTGRCeGTTTTTTRTRTRTTTGTT~TRRRRTRRRCRTRRRTRRTR~ReCRRR~TRe~GGCT~T tRNA

Ile

.,~oo

,z,o

.....

TRGCT~GTTGGTTR~R~CGTTG~TTT~TRRGG~R~R~GTCGR~R~TTCRR~TCTTTC~TRGC~CRcCTTCR~TTTRC~RR~TT~R .

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I

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1410

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TCCGCTTRT~TCc~C~RTGCCnG~TRTTnRRTTTRRRTTTTRRTTTRnnTTT~TTTT~cTCRRRn~GRCGTCCcCTTRCG~GRT~Tc~

~TGTTRGTGGCRGTGGCCTGcRcTGCGRRTTTRTTRGcCGTRGGC~R~GCcRGCTGRT~TTT~TR~TCGcTG~TTTG~RGG~GRRGG~G~ .~seo

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RGRTTTTR~TGCT~C~FTRRR~TTTRGTTRCC~RRTRTTTRTRTTRG~RT~TTRRTRCRRTRRRTRRRTTTGTTG~R~GC~R~RRRTTT

~TTTRTTGTRTR~RRRT~TCCRCTn~TnTTT~T~TCCCGTR~GGGG~GC~G~RG~GRRGGGGTTT~T~G~T~TRTRTRCCRTR

GC~GGTRG~RCTRRRT~TTRRGRGCGRR~C~RRT~GeRRRR~RRR~RRRCT~cT~CTRRRGRGGT~R~T~TTTTeTRR~

CRRRT

Fig. 2. Nucleotide sequence of the non-coding strand of the entire 1 6 S - 7S ribosomal spacer. The 3' end of the 16S rRNA gene immediately precedes base 1 and the 7S rRNA gene starts at base 1806. The genes of tRNAile and tRNAala are framed. IR7 and IR2 are two nearly perfect inverted repeats (cf. Figs. 4, 5).

267 and the sequencing strategy used. Both strands were sequenced except for a small 14 bp region where only one strand was sequenced three times independently. The 1 805 bp sequence of the noncoding strand of this spacer is shown in Fig. 2. Previous work has shown that the ribosomal spacer of C. reinhardii hybridizes with total cellular 4S RNA (12). Screening for GTTC allowed us to localize two t R N A genes which code for t R N A ala and t R N A ile as judged by their anticodons. These two genes are, respectively, 90 and 72% homologous to their maize counterparts (8) and 97 and 82O7o homologous to their Euglena counterparts (6, 18). The cloverleaf structures of these tRNAs are shown in Fig. 3. As observed for other chloroplast t R N A genes, the 3 ' terminal CCA of the mature tRNAs is not encoded by chloroplast DNA, but added post-transcriptionnally. We have recently shown that these two t R N A genes are functional by demonstrating that the chloroplast t R N A ile from C. reinhardii and t R N A ala from pea hybridize exclusively to the chloroplast EcoRI fragment containing the ribosomal spacer (2). Although the chloroplast ribosomal spacer is highly conserved among higher plants, no sequence homology is apparent between the ribosomal spacers of C. reinhardii, maize, Euglena or E. coli except for the two t R N A genes. A striking feature of the C reinhardii spacer is the presence of numerous short direct and inverted repeats of at least 15 contiguous nucleotides. The arrangement of these structures is displayed in Fig. 4. As shown in Fig. 5 several of these repeated elements can be folded into a large palindromic structure which occupies nearly a third of the spacer (positions 273 and 842 in Fig. 2). Because of the presence of other inverted repeats (IR1, IR3) this folding does not necessarily Au t-RNA I l e

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Fig. 3. Secondary structures of tRNAile and tRNAala. Differences with the corresponding tRNAs of Euglena (6, 18) are indicated by smaller letters.

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Fig. 4. Location of inverted (IR) or direct (D) repeats in the ribosomal spacer. The lower part of the figure shows the arrangement of overlapping repeated elements in more detail. With reference to Fig. 2, the positions of these repeats are: IRI (221-253, 430-392, 592-614, 724-683); IR2 (346-451, 658-553); IR3 (168-183, 201-219, 483-504, 934-911, 1582- 1605, 1631- 1611); IR4 (901-955, 1613- 1561); IR5 (200- 228, 1629- 1601); IR6 (872- 840, 1421- 1450, 1669-1640); IR7 (273-337, 842-778); D1 (277-300, 503 - 531). represent the only possible secondary structure of the ribosomal spacer. However it should be noted that the extensive secondary structure displayed in Fig. 5 is compatible with the structure of the singlestranded spacer observed in the electron microscope (22). It is noteworthy that this structure includes a region containing the first half of IR2 (Fig. 5) which is complementary over 51 bases to a D N A segment on the 5' side of the 16S r R N A gene as shown in Fig. 6. It can also be seen that a second 49 bp pairing occurs between two other 5' and 3' flanking regions. Complementary regions that flank the 16S r R N A gene are also found in chloroplasts from higher plants (26) and in E. coli (3). They appear to play an important role in the folding and in the maturation of the r R N A precursor. It has been shown that the chloroplast r R N ~ genes of higher plants and of Euglena are tran scribed into a large precursor which is processe( into mature rRNAs (1, 27). We have been unable tt detect chloroplast rRNA precursors in C. reinhardi using spacer D N A fragments as hybridization probes, presumably because processing closely follows the transcription of the ribosomal unit. Small repeats of the same type as those of the ribosomal spacer also occur in other non codingregions of the chloroplast inverted repeat of C. rein-

268

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A-T A-T T-A A-T A-T A-T T-A T-A T°A T-A A-T G-C T-A G-C G-C • C-G A-T G-C T-A T-A G-C C-G C-G T-A C-G G-C C-G C-G T-A A-T T-A C-G G-C G-C C-G T-A A-T A-T C-G G A-T

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Fig. 5. Large palindromic structure in the ribosomal spacer• The structure has been cut in half (large arrows). IRI, IR2, IR3, IR7 and D1 are defined in Fig. 4. Numbered nucleotides are as given in Fig. 2.

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171 bp

(5) as suggested by the observation that a 1.5 kb fragment within the inverted repeat hybridizes to numerous chloroplast DNA regions (21). The function and the origin of these repeated elements is still unknown. They could possibly be the remnants of chloroplast transposable elements, and they may have some role in chloroplast RNA processing, intramolecular recombination (within the inverted repeat) and chloroplast DNA folding.

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Acknowledgements

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We thank O. Jenni for drawings and photography and J. Erickson for helpful comments. This work was supported by grant 3.587-0.84 from the Swiss National Foundation.

bp = IR 7

A

T

T

GATA

References

CG 51 bp TA GC T T 5 '. . . . . A A A T

CC . . . . . .

3'

Fig. 6. Folding of 5' and 3' flanking regions of the chloroplast 16S rRNA gene from C. reinhardii. The 5' and 3' ends of the 16S rRNA gene (4) are indicated by arrows. IR7 is defined in Fig. 2. Stem structures of 49 and 51 bp are indicated by brackets. The entire 5' flanking sequence will be published elsewhere.

hardii (Schneider, Erickson, Darlix and Rochaix, in preparation). It is possible that these direct and inverted repeats may be the cause of deletions and inversions which have been observed in the chloroplast inverted repeat of C. reinhardii (17, 19). Palmer et al. (19) have recently examined the chloroplast ribosomal unit of C. smithii which is interfertile with C. reinhardii. The ribosomal units of these two species appear to be very similar except for several deletions and insertions. It is interesting to note that the ribosomal spacer of C. smithii contains an insertion which maps within or very near the large palindromic sequence of Fig. 5 (19). It is possible that the observed D N A rearrangements may be due to recombination between repeated elements or to amplification of these elements. These repeated elements could belong to the family of short inverted repeats which are scattered throughout the chloroplast genome of C. reinhardii

1. Bohnert H J, Driesel AJ, Herrmann RG: Characterization of the RNA compounds synthesized by isolated chloroplasts. In: Bucher T, Neupert W, Sebald W, Werner S (eds) Genetics and biogenesis Of chloroplasts and mitochondria. North-Holland Biomedical Press, Amsterdam, 1976, pp 629-636. 2. Bergmann P, Schneider M, Burkard G, Weil JH, Rochaix JD: Transfer RNA gene mapping studies on chloroplast DNA from Chlamydomonas reinhardii. Plant Science 39:133- 140, 1985. 3. Brosius J, Dull TJ, Sleeter DD, Noller HF: Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J Mol Biol 148:107-127, 1981. 4. Dron M, Rahire M, Rochaix JD: Sequence of the chloroplast 16S rRNA gene and its surrounding regions of Chlamydomonas reinhardii. Nucl Acids Res 10:7609- 7620, 1982. 5. Gelvin SB, Howell SH: Small repeated sequences in the chloroplast genome of Chlamydomonas reinhardii. Mol Gen Genet 173:315-322, 1979. 6. Graf L, K6ssel H, Stutz E: Sequencing of 16S - 23S spacer in a ribosomal RNA operon of Euglena gracilis chloroplast DNA reveals two tRNA genes. Nature 286:908- 910, 1980. 7. Katz L, Kingsbury DT, Helinski DR: Stimulation by cyclic adenosine monophosphate of plasmid deoxyribonucleic acid replication and catabolite repression of the plasmid deoxyribonucleic acid-protein relaxation complex. J Bacteriol 114:577-591, 1973. 8. Koch W, Edwards K, K6ssel H: Sequencing of the 16S- 23S spacer in a ribosomal RNA operon of Zea mays chloroplast DNA. Cell 25:203-213, 1981. 9. Kuntz M, Crouse E J, Mubumbila M, Burkard G, Weil JH, Bohnert H J, Mucke H, L6ffelhardt W: Transfer RNA gene mapping studies on cyanelle DNA from Cyanophora paradoxe. Mol Gen Genet 194:508-512, 1984.

270 10. Loughney K, Lund E, Dahlberg JE: tRNA genes are found between the 16S and 23S rRNA genes in Bacillus subtilis. Nucleic Acids Res 10:1607- 1624, 1982. 11. Lund E, Dahlberg JE, Lindahl L, Jaskunas R, Dennis PP, Nomura M: Transfer RNA genes between 16S and 23S rRNA genes in rRNA. Transcription units of E. coli. Cell 7:165- 177, 1976. 12. Malnoe PM, Rochaix JD: Localization of 4S RNA genes on the chloroplast genome of Chlamydomonas reinhardii. Mol Gen Genet 166:269-275, 1978. 13. Maxam AM, Gilbert W: Sequencing end-labeled DNA with base-specific chemical cleavages. In: Methods in Enzymol 65:499- 560, 1980. 14. Mubumbila M, Gordon KHJ, Crouse E J, Burkard G, Weil JH: Construction of the physical map of the chloroplast DNA of Phaseolus vulgaris and localization of ribosomal and transfer RNA genes. Gene 21:257-266, 1983. 15. Mubumbila M, Crouse E J, Weil JH: Transfer RNAs and tRNA genes of Vicia faba chloroplasts. Current Genet8:379- 385, 1984. 16. Mubumbila M, Bowman CM, Droog F, Dyer T, Kuntz M, Weil JH: Chloroplast transfer RNAs and tRNA genes of wheat. Plant Molec Biol 4:315-320, 1985. 17. Myers M, Grant DM, Robert DK, Harris EH, Boynton JE, Gillham NW: Mutants of Chlamydornonas reinhardii with physical alteration in their chloroplast DNA. Plasmid 7:133- 151, 1982. 18. Orozco EM, Rushlow KE, Dodd JR, Hallick RB: Euglena gracilis ribosomal RNA transcription units. II Nucleotide sequence homology between the 16S- 23S ribosomal RNA spacer and the 16S ribosomal RNA leader regions. J Biol Chem 255:10997 - 11003. 19. Palmer JD, Boynton JE, Gillham NW, Harris EH: Evolution and recombination of the large inverted repeat in Chlarnydomonas chloroplast DNA In: Arntzen C J,

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26.

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28.

Bogord L, Bonitz S, Steinback K (eds) Molecular Biology of the photosynthetic apparatus. Cold Spring Harbor, in press. Rochaix JD: Restriction endonuclease map of the chloroplast DNA of Chlamydomonas reinhardii. J Mol Biol 126:597-617, 1978. Rochaix JD: Organization, function and expression of the chloroplast DNA of Chlamydomonas reinhardii. Experientia 37:323 - 332, 1981. Rochaix JD, Malnoe PM: Anatomy of the chloroplast ribosomal DNA of Chlamydomonas reinhardii. Cell 15:661 - 670, 1978. Schwarz Z, K6ssel H: The primary structure of 16S rDNA from Zea mays chloroplast is homologous to E. coli 16S rRNA. Nature 283:738- 742. Takaiwa F, Sugiura M: Nucleotide sequence of the 16S-23S spacer region in an rRNA gene cluster from tobacco chloroplast DNA. Nucl Acids Res 10:2665- 2675, 1982. Tohdoh W, Sugiura M: The complete nucleotide sequence of a 16S ribosomal RNA gene from tobacco chloroplasts. Gene 17:213-218, 1982. Whitfeld PR, Bottomley W: Organization and structure of chloroplast genes. Ann Rev Plant Physiol 34:279-310, 1983. Wollgiehn R, Parthier B: RNA synthesis in isolated chloroplasts of Euglena gracilis. Plant Sci Lett 16:203-210, 1979. Young RA, Macklis R, Steitz JA: Sequence of the 16S-23S spacer region in two ribosomal RNA operons of Escherichia coli. J Biol Chem 254:3264-3271, 1979.

Received 20 September 1985; in revised form 10 December 1985; accepted 16 December 1985.

Sequence organization of the chloroplast ribosomal spacer of Chlamydomonas reinhardii: uninterrupted tRNAile and tRNAala genes and extensive secondary structure.

The 1805 bp spacer between the chloroplast ribosomal 16S and 7S RNA genes of Chlamydomonas reinhardii has been sequenced. It contains the genes of tRN...
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