Sequence organization of the chloroplast ribosomal spacer of <i>Chlamydomonas reinhardii</i>: uninterrupted tRNAile and tRNAala genes and extensive secondary structure

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Sequence organization of the chloroplast ribosomal spacer of Chlamydomonas reinhardii : uninterrupted tRNAile and tRNAala

genes and extensive secondary structure

SCHNEIDER, Marc, ROCHAIX, Jean-David

Abstract

The 1805 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.

SCHNEIDER, Marc, ROCHAIX, Jean-David. Sequence organization of the chloroplast ribosomal spacer of Chlamydomonas reinhardii : uninterrupted tRNAile and tRNAala genes and

extensive secondary structure. Plant Molecular Biology , 1986, vol. 6, no. 4, p. 265-270

DOI : 10.1007/BF00015232

Available at:

http://archive-ouverte.unige.ch/unige:151548

Disclaimer: layout of this document may differ from the published version.

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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 rein- hardii 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

In several higher plants and algae the chloroplast ribosomal units are located within a large inverted repeat of the chloroplast genome (26). In C. rein- hardii 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-

* Paper presented at the First International Congress of Plant

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 compara- ble to that of higher plants and since it is known to hybridize with 4S RNA (12), it was of interest to ex- amine 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.

Materials and methods

The recombinant plasmid HRI.14 containing most of the chloroplast ribosomal spacer of C.

reinhardii has been described (22). Plasmid DNA

Molecular Biology (Savannah, GA, 1985).

265

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266

was prepared by the method of Katz et al. (7). DNA fragments were 3' or 5 '-endlabelled and sequenced by the chemical cleavage method as described (13).

The DNA sequence analysis was performed on a Hewlett Packard computer, model 9845.

Results and discussion

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

i•

^{BB mm}^lle^Ato ^7S ^[r

l t :T_-';---

TTT TIT

100bp

Fig. 1. Restriction map and sequencing strategy of the chlo- roplast ribosomal spacer of C. reinhardii. The genes of tRNAile 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 .leo

~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

• ~,o t~Y. .,~o .,,o

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

.--o IR2 .,,o ^.,,o

~ G~A~CTGC~TRGGCRRGRRR~TTRGGG~TTTTRRTG~RRTRRRTRRRTTTGTC~CCTTCGGGTRRRTRRRTTTTRGTGGRcGCCRGTG

.$70 .600 .e3o

GRTRTTTRTRT~RGGR~GTcRsTGG~RGTGGTRCCG~RCTG~TGCR~GCC~TC~TT~GGRGRTGTR~R~C~TT~GG~AA~TRRRGTT

.--o IR2 .o.o ^....

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

.750 .780 .s~o

~gGCR~nRTTTRRTTnRTTG~E~GTRRGG~G~GG~RTRTT~TnTRCC~G~RG~R~CTGGTTRG~RTRGGE~RRCTG

.~,o .-,o I R 7 ^.~oo

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

.gad .g~O .ggo

~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

. . . . . . . . . tRNA AIo .... o

~T~RTcR~T~TTRRT~RCR~TTTRRRR~GGRTRT~G¢TCRGT~GGTR~RG~G~TGCCTT~GCRRGGCRGRTGTCRGCGGTTCG~

.13BO 1410 .l~ao

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

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

.~seo ~5~0 .~e~O

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 16S- 7S ribosomal spacer. The 3' end of the 16S rRNA gene im- mediately 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).

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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 non- coding 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 tRNA genes which code for tRNA ala and tRNA 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 tRNA genes, the 3' terminal CCA of the mature tRNAs is not encoded by chloroplast DNA, but ad- ded post-transcriptionnally. We have recently shown that these two tRNA genes are functional by demonstrating that the chloroplast tRNA ile from C. reinhardii and tRNA ala from pea hybridize ex- clusively 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 tRNA 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 A

G-C G C

GC G-C

t-RNA Ile G C I-RNA AtQ G-U

C-G G C

U-A G GA-U

G A-U o U A G

U U ACUCGAuU AC~CjucUAA uUGAcucGAuA-UucGccUAA

GO GAGCG _{UU A} AU_AA CCU UU G~AL~ C A G GAG~2 GuA UC_GA CU ~GCGG G UU ~

AU Au GAGU U A U G

cG CG G C

~. C -G C-G

d

^{U G A} ^U^{C G}^A

UoAuA UuGcA

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

16S

IRI I)

JR2 [ P3 EW) IRZ.

ZR% I) JR6

[p7 I )

(I b (I ) ( I

I)

ALQ 7S{

~m ,J

(I !)(i

I) (I

(I

I)

; f

(I ( i

I) (I

, --4 [~1; ~:~ ~ ~j ~ , ~ .~

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 single- stranded spacer observed in the electron micro- scope (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 DNA segment on the 5' side of the 16S rRNA 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 rRNA 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 rRNA precursor. It has been shown that the chloroplast rRN~

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 DNA fragments as hybridization probes, presumably because processing closely fol- lows the transcription of the ribosomal unit.

Small repeats of the same type as those of the ribosomal spacer also occur in other non coding- regions of the chloroplast inverted repeat of C. rein-

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268

5 ' - - I R I ---oA

IR7

CT . . . 3' _ A A --

273.G-C.842 G-C A-T .G-C C-G T-A C-G C-G C-G C-G T-A T-A C-C G-C G-C G-C

c-G 01

A A-T

A-T T-A A-T A-T T-A A-T 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 G-C C-G 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 A-T G AG_cC

T-A T-A C-G C-G

;38.T-A.778

GCGT GGCCCATATATTATACC AGTA CTCGTAAATTTATTTG C

T-A.658 T

A-T G

T-A C

AA_TA TA_TT T-A A-T C-C C-C A-T T-A G-C T-A T-A ~-T A-T A-T C-G T-A I;-C

#

C-G

GTTAATTAAT

G T

C T

C A

G A

T A

A C

G A

G

G -G

C G

C ¢

G C

G G T T G A

~ IR1 G C

c G

A A

G G

C G

A A

G G

G A G G ATACTCCGA A

IR2

G-C A-T T-A A-T A-T A-T C-G T-A T-A T-A A-T G-C T-A T-A C G-C

C-G C-G G-C A~T A~T

IR1

T-A A-T C'-'G

C"'G A-T C-G T-A G-C C-G C-G A-T C-G T-A G-C G T

C-G G-C T-A C-G 151 ,T-A. 553 C-G GGATTCAAAGAACGG ATC CGGTC AACCG TT&TATTTAT&GGTG

A , g

T ""1 IR3 Ol c

T I A

AA TGC AAT AA AT AA A 1%*~ G T C C C C TT CG G G TA .=U=.T AA ATI~'F~ AGTGG

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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.--16 S RNA~

5'16 S C t~ ~X

TCCA GG T I

A T AGATTTGA J

A TTTAAATT i t

I GAG AA T T t~--3'16 S

171 bp ~\ • 178 bp

"A A A A'

A G AT AT GC GC

49 bp : :

GC A C

GC T T C T

TC T C C T T C G G A G T A A AG G A AGC C T e A T A A

TATG TC~G T A T T

AATA T A TT

TA AT

TA 65 bp = IR 7 A A

T T

GATA CG 51 bp

TA GC T T

5 ' ... AAAT 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 brack- ets. The entire 5' flanking sequence will be published elsewhere.

hardii (Schneider, Erickson, Darlix and Rochaix, in preparation). It is possible that these direct and in- verted repeats may be the cause of deletions and in- versions which have been observed in the chlo- roplast inverted repeat of C. reinhardii (17, 19).

Palmer et al. (19) have recently examined the chlo- roplast ribosomal unit of C. smithii which is inter- fertile 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 con- tains an insertion which maps within or very near the large palindromic sequence of Fig. 5 (19). It is possible that the observed DNA rearrangements may be due to recombination between repeated ele- ments or to amplification of these elements.

These repeated elements could belong to the fa- mily of short inverted repeats which are scattered throughout the chloroplast genome of C. reinhardii

(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, in- tramolecular recombination (within the inverted repeat) and chloroplast DNA folding.

Acknowledgements

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

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