The complete nucleotide sequence of a 23S rRNA gene from a blue-green alga, Anacystis nidulans

Gene, 24 ( 1983) 2 19-225

119

Elsevier GENE

838

The complete nucleotide sequence of a 23s rRNA gene from a blue-green alga, Anacystis ni&~lans (Cyanobacteria;

chloroplast

23s rRNA;

sequence

homology)

Masanobu Kumano, Noboru Tomioka * and Masahiro Sugiura ** Dainippvn Ink and Chemicals, Inc., Nihonhushi. Chuo-ku, Tokvo 103, Tel. (03) 272-4511: * Biotechrzology Luhorator!: MitsuiTotrtsu Chemicals, Inc., Mobara, Chibu-ken 297, Tel. (04752)4-1169. and ** Department of Biology, NagoJw Univrrsit~. Chikusrr-ku. Nagoya 464, Tel. (052) 781-51 I I (Japan) (Received

April 1st. 1983)

(Accepted

May 18th, 1983)

SUMMARY

The complete nucleotide sequence of a 23s rRNA gene from a blue-green alga, Anact~stis niduluru, has been determined. This nucleotide sequence has 78 y0 and 68% homologies with those of the tobacco chloroplast and Escherichia coli 23s rRNA genes, respectively. The 3’-terminal region of the A. nidulans 23s rRNA gene has strong homology

with the chloroplast

4.5s rRNA.

INTRODIJCTION

Blue-green algae (Cyanobacteria) are autotrophic prokaryotes which carry out photosynthesis. It is of interest to compare blue-green algal genes with chloroplast genes because of the endosymbiotic theory that chloroplasts were derived from an ancestral photosynthetic prokaryote related to bluegreen algae. We have extensively sequenced the rRNA genes from tobacco chloroplasts (Takaiwa and Sugiura, 1980; Tohdoh et al., 198 1; Tohdoh and Sugiura, 1982; Takaiwa and Sugiura, 1982a,b) and have cloned the rRNA gene clusters from A. niduluns 6301 (Tomioka et al., 1981). Here we present the

** To whom all correspondence

and reprint

requests

should be

addressed. Abbreviations:

bp, base pairs;

kb, kilobase

pairs;

rRNA,

somttl RNA.

0378-l 110,83W~3.013 0

1983 Elsevier

Science

Publishers

ribo-

complete nucleotide sequence of the A. uidukans 23s rRNA gene and demonstrate strong homology with that of the tobacco chloroplast 23s rRNA gene.

MATERIALS

AND

METHODS

Recombinant plasmid pAN4, which contains one copy each of the genes for the 16S, 23s and 55 rRNA sequences (Tomioka et al., 19x1), was used. The plasmid DNA was digested with Pst I + Hin dII1, or PstI + Kpn I + SstII. The digests were separated by 5% polyacrylamide gel electrophoresis. The DNA fragments containing the 23s rRNA sequence were recovered from the excised gel pieces by electrophoresis and purified by DEAESephacel column chromatography (Tohdoh and Sugiura, 1982). Sequencing of these fragments was performed according to the method of Maxam and Gilbert ( 1977).

RESULTS

AND

The 6.5kb

68”/ sequence homologies with those of tobacco chloroplast and E. cob, respectively (see Fig. 2).

DISCUSSION

and 8.0-kb PstI fragments

A. nidulam rRNA gene clusters pBR322

(Tomioka

et al., 1981). The rRNA

cIuster located in the 6.5kb

6.5kb

plasmid

PstI fragment,

gene

fragment was designated

as rm4 and that in the 8.0-kb Recombinant

coding for

have been cloned in

fragment

as rmB.

A. nidulans 16s rRNA gene has been sequenced found to be 83’::, and 74”,, homologous tobacco

chloroplast

and

(Tomioka

and Sugiura,

1983). A. ~~~~~~~~~~ 5s rRNA

has 697, and 60T0 homologies

contains

the

chloroplast

was used for sequencing

the

Bowman,

pAN4,

which

and

to those of

and

E. CO/~, respectively with those of tobacco

E. coli, respectively

1979; Ifori

and Osawa,

(Dyer

and

1979; Takaiwa

23s rRNA gene. A physical map and the sequencing

and Sugiura,

strategy for the 23 S rRNA gene are shown in Fig. 1.

the 23s rRNA genes is less than that among the 16s

Most of the sequences both

DNA

strands

were confirmed

by sequencing

rRNA

and more than

twice in each

rRNAs.

direction. The complete nucleotide sequence (RNAIikc strand) of the A. ~~~~~~~~ 23 S rRNA gene in UI?A is shown in Fig. 2. The 5’ and 3’ ends of the 235 rRNA-coding region were deduced on the basis of homology with the E. co& 235 rRNA gene (Brosius et al., 198 1). The coding region of the A. nidu/ans 23s rRNA gene is thus tentatively estimated at 2869 bp, which is 35 bp shorter than the E, co/i 23s rRNA gene. ‘The .4. niduluns 23s rRNA

gene has 789,, and

16 S

genes,

198 1). The degree of homology but more

than

that

among

among the 5S

Chloroplast 4.5s rRNAs are known to have homology with the 3 ‘-terminal region (positions 2808-2902) of E. cttfi 23s rRNA (~~achatt et al.. 1981). This region of E. ccl/i 23S rRNA has 56’i,>, 57”, and 57P;, sequence homologies with the 4.55 rRNAs of tobacco (Takaiwa and Sugiura, 1980) wheat (Wildeman and Nazar, 1980) and maize (Edwards et al., 1981), respectively. The 3’-terminal region (positions 2775-2867) of the A. niduhs 23s rRNA gene shows high homology with the chlnro-

221

ATAGAGATGAGAGTGTAGGTATCACAGACACCATCTTCATCATCACTTGATT~AGTCAAGTGGGAGAAATGGAAGTCAGAAAGAAGTAGT 5’end ,

~23s

.,..G..T...C.TAC.,......,GC,..~,,..GT....,.,.,T.,.....,.~....AT.TG,..,.A,,G....TA,,G,,AT,~G,.C

t23S

AGCTACGAAGGGCTTACGGTfFAT

CTAECCACACAGAG~CGAAGAAGGACGTGGCT~CGACGATACGCCTCGGGGAGCTGGAAG

,A,G,G.,.A.............. ,5’end

..,.....C,....A,.,G,....G.,.A.~..PT.....,A.T..T....,...T,.A..A

.G.,~T.A...GC..,............A.,...AG.,TGTTTCGACACAC,.TCATT.A..........,..T.AA~.,.G......G,.G~~~C., CATflATCCGAI'GATTTCCGAATGGGGCAACCCCATG

194 181

~TG..,.,.,..,G..cA.,.AA...A.~~....T.,..,.c,.

i79

~~~~2..i.:,..;..T..Cr.:i.....:.........

283

~GCCGGAGCAAGAGAAAACAAAAGTGATTCCCTCAGTA(;CGGCGAGCGAACGGGGACCAGCCTAAACCAAACTCCACGGAGTTTGGG

: ,,.,

A

,,,.,,

A

,,.,,

96 9F 95

TACGGCCACCTGAATCCATAGGGTGGCGCGAfiGAACCCGGCGfiATTG

~.~...,..GA....C....,,A.,......

,.,,**,...,,

-1

G......C,,,,...GT,.,,....,.......AT..,,G...........GTGAAAn,,.,~...GT..

280 277 359 378 373

..,.,.GG..,GA....,.GT.,C.T..C.....AT.C.G......T...,~C......,..........AC............... GAGTAGCACGGAGCACGTGAAATTCCGTGTGAfiTCCGCLAGGACC .CTGA.....,.,,,.T..G,,,,.,.G.,,C.,.........GA,k,.....

455

CTCGTLAGGCTAAATACTCCT~TGTGACCGAAC

475

..T.C.....,.......,,..G.......,,..C.,.G

468

,1,11,,1 T ,,,II.,,l, C ,......,..,.## CG......,,.,,.A,....C,......,..TA.G....,...,,G....,A..C..AGG..T...

555

CAGTACCGCGAGGGAAAGGTGAbAAGAACCCC

~GGAGTGAAATAGAACATGPAACCGTGAGCTTACAPGCAGTCGGI~GCCCGATTCAACGGGTG

574

T ,,,,..,T ,,,#,,,G ,,,,,,,*,,,,,,,

. . . . .. . . . . . . . .. . .. . .. . .. ..G....CC........G....G..A(

562

.I T ,,,,

A..,T...T.T..,.G.T.A.

GGcrc..

. .. . . . ..TT.TG.P....A......~.C...T~~.G~.G....G...C....C.......T..CT,..,.T

652

ACEGCSTCCCTGTTGAAGAATGAGCCGGCGACTTATAGGCACTGGCA~GTlAAGGCGGAAAT~CCGPAGCCAAAGCGf\AAGCGAGTCTGAATA~G(,CG~~

673

,,C ,,,,,,.,,I,.,.,,,,l. ..,.,,,.,. C ,,,,.,, G ,,,,TT,,.....,(1,A.CCCA...G....GT.....,......,,.TC.......A.

661

..A..TGCA.GG..........A.,...~,.,......G.,,...G...,T.,...G.,,........T..C....,.A,.......,.T~\,...,....

751

ACCCGSGTGATCTAACCATGGCCACGATGAkGCTTGGEiAACACCAAGTGGAGGTCCGAACCGACCG~~TGTTGA~A

771

. ..T.........,T.....A..,..,,,,,,..,....G.A..T......,...,......,,,T,..,,..,.6

759

I. rA....,.,,CT...,.C.G..,..,..,G,.,....A,.,,G...,A.,.,,..,.,.........~.TA,,T,,,T~,...CC

847

AATCGI;CGGATFAGCTGTGGTTACGGGGTGAAATGCCAATCGAACCCGGAGCTAGCTGGTTCTCCCCGAAATA~TTG~!GGCGT,~GCGGT

867 856

C.,CT.~G..,,...,,..,..T.....C TAGrGGTGGGGTAGAGCACTGATTCGGT ?CTf--jA

,,,#..A 111111,T ,I,,,,

G...T.AT.CC..~T,.....CC..,TG.....,~.,.....~.G.AGAATG

933

TGCGAGAGCGGTACCAAATCGKTCAAACTCCLPATACGCCG .C...,..................G.......T,.,...TAGA.

950 .GACCTCAAAATA..

949

..C.GG..A..C..G.C...T,C...CG.,,G.C.,G...,,,,.,,.,A,.,.,...G...,...,..,,.C6..G...TGGT.A....

1024

CCATGCCAGTCAGkCTGTGGGGGATAAGCTCCATGGTCAAGAGGGAAACAGCCC~\GACCACCAGCTAAGGTCCTCAAATCAGAACTT~~GT~

1041

GGGGTCAAGGTCG,.T...G....GA,......,.....T...C...G..,............G..T.,..,.......C,.CT...,G,TCG.,C...,

1049

GG .,.C.,T:,.. .,AG..CC.,...G......T...G..T,.,.......,..,A.,T....,AA............,..,,.,G..T..GC.~..... ’

1123

f\TAPAGGAGGTGGGAGTGCATAGACAACCAGGAGGTTTGCCTAGAAGCAGCCATCCTTAAAAGAGTGCGTAATAGCTCACTGGTCAA

,

I..

1140 1148

A..G.....G.....G................,,..,....,C.,..G...........,........,..A.,G.

G * I.G,.,T........,,.ACCA..C........,,C..C.GC,ACGCT.A...GTTG..G............,..T..~ ACGGGGCTAAGTTCTGTACCGAAGCTGTGGAATTG CGA.C.G.., m.,..,...G..GT

..CC..~,AA.GT.T.

ATfiGTAGGGGAGCGTTCCGTCGTAGGGTGAAGCGGTAGCG .C,......,..,.....~~CC.T,fl.A.~~

1220 Ii32 1233 1313 1322

CCTC.GIk@if...[

GTG..,,.....G......C...................A..C......T..,,.......A.......,...A..T.,...,.

1331

,,.GTCC.,C.T.AA.,G.G.C.....G.,..GACC,,..,........C..,AG.,.,.........GA.,,.................,T CCTCCGGAAGGCTCGTCCGCGGAGGGrTACTCAGGTCCTAAGGC~~\GGCAC~~A~TGCGTAGTCC~~TG~ACA~CAG[;TT~\~\T~\TTCCTGTACC ,,,.,. C .I..T......A....,,.,6,,..,,.G,,...,FTC.,..C,..AG..TA[1.....,.,.........G...........,.TGCCCCT.G

, , , ,TCC~,,.,,,,,,.,6,,,,,,,,TT,..i:,i;.::,;;.,,...T,~,C-,T,,.GARCGTACiG~TGTCCCTGC

223

kGT.,,,.,.T,A..,...,T,GG...G,.A..,...T,GC.CT~..CC,....,..AA,.C.

2764

CGCGAAGGACGCACCGCTGGTGTACCAGTTATCGTGCCAAC~~AACGCTGG~TaGCTACGTGTG

,A.U....GT.,.,.....,,.,...CAC.. GTEGATAACCGCTGAAAGCATCTAAGTGGGA

2737

I,...,,.I,.I,.I T
.C.,,.,,.T.,....,...,,.,...A,T.

2777

5

.A.TTG,C.CG........T,.,C,TGAC I AGCCCACTCBAGATGAGTACTCTCATGGCA

2797

CTT

2767 GACTTCCCCAGAGCCTCCG~TAGCACAGCCGAGACAGCGACG~GTTCTCTGCCCCTGCGGGGATGGAG Sp,acer( 101 t?pl

2877 2870

CGGG,,..T...CG,...G...CG.TG..

c ,#.,,,

2836

AATACGCGCTATGTGGAAGTTCAGCAATGGATGAAGC

2976

TATCATTACG..,..T.TC.A........G..,TG.,,T...C... 4.5 S

rDNA

(103bp) 2904 2869 3008 2901

TAACACTT-GATATCGSCACTCTCCTCTATGC

Fig. 2. Nucleotide comparison. indicate

nuclcotides

in the row. relative proper

sequence

sequences

ofthe A.

nidulms

23s rRNA

gene. Only the noncoding

ofthe E. co/i (top row, E) and the tobacco

identical

with the A. nidulnm

sequence.

to the 5’ end of each 23s rRNA-coding

chloroplast

The arrows

(RNA-like)

(bottom

represent

region. The notches

strand

is shown (middle

rev,, A). For

row, T) 23s rRNA genes are presented;

inverted in framing

repeats. represent

The numbers deleted

the dots

refer to the last bp

nucleotides

as to assure

alignment.

23SrRNA ‘,

,’ plast 4.5s rRNA sequences. The degrees of homology with this region are 68%, 71?;, and 69% with the 4.5s rRNAs of tobacco, wheat and maize, respectively. These homologies are higher than those with the E. coli region. The 23s and 4.5s rRNA-coding regions of tobacco chloroplasts are separated by a lOl-bp spacer. Little homology with this region was found between the A. niduluns and E. coli 23 S rRNA genes. Moreover, the sequence (positions 2764-2774) in the A. niduians 23s rRNA gene corresponding to the chloroplast lOl-bp spacer has weak homology with that (positions 2792-2807) in the E. coli 23s rRNA gene. This region therefore seems to have been variable during the course of evolution. These sequences contain short inverted repeats, as shown in Fig. 2. The meaning of these inverted repeats is not known at present. In E. co/i the complementary structure flanking the 23s rRNA is known to be necessary for processing of its rRNA precursor (Bram et al., 1980). A similar arrangement was found in sequences surrounding the 5’ end of the 23s rRNA gene and the 3’ end of the 4.5s rRNA gene in the tobacco chloroplast (Takaiwa and Sugiura, 1982b). A secondary structure for the regions surrounding the 5’ and 3’ ends ofA. nidulans 23s rRNA can be constructed, as shown in Fig. 3. This suggests that A. nidulans has an rRNA processing mechanism similar to E. coli.

‘“A

CG~’

U C-G. G-C A-U A-U C-G U-A G-C $G-

C u

u

A’UC G “u

it

c

LJ

A-U C-G U-A .A- u U-

A

G U G-C A G u G G-C J-A G-C A-U, .G-C A-” G-C II

+,A

3

5’

Fig. 3. Possible rounding quence.

secondary

structure

c

-A U G-C A-U G A A-U u G ;2900

for the

sequences

the 5’ and 3’ ends of the .4. nidulcrns 23s rRN.4 The arrows

indicate

sites of processing.

surse-

rRNA interact with tRNA,?““. Homologous regions in tobacco chloroplast and A. nidularzs23s rRNAs have 7 nucleotides (positions 1998-2005 and 1967-1974, respectively) iilt~racting with tRNhfh’IC’ (Kamogashira, T. and Sugiura, M., unpublished ; Ecarot-Charrier and Cedergren, 1976) as shown in Fig. 5. A. ~jd~~a~~235 rRNA is thus more similar to tobacco chloropiast 235 rRNA in this region than to that of E. e&i.

Branlant et al. (198 1) postulated a general structure for the protein Ll binding region (positions 2091-2198) of bacterial 235 rRNA. A similar secondary structure coufd be constructed from the corresponding region (positions 2103-2203) of tobacco chloroplast 23s rRNA (Takaiwa and Sugillra, 1982b). A. ~idl~~~ns23s rRNA also contains a homologous structure, as shown in Fig. 4. Dahlberg et al. (1978) proposed a mode1 in which 17 nucleotides (positions 1984-2001) of E. coli 23s

U.G U-A.

G

AAG-Cc

G-C *

GAwU~

AG

GA

G-C

AG-Ckc~Gu G

A~-G~&CUU G G-CAtJA+G

AG-CA‘ACGGu

E-:AufiGAG

$-;G AGGAU U-2180 UGUGUA- U

AGcUU ‘tCGUC

A-U

C - G&) C-G U-A U-A

AUAG’

‘C

AGG

GGCWG_GU hi2160 G-c U-i U-A U-A

2080-g

2100-G- u U-G*

G

GG_CAUAGA G-C * ,U-A G-C

UIA .G-C AG, U

;I$

G

*UAIJC_G~

;

G- U F

U-G

A

U - Ah

5’

C”

5’

‘A

Gc

G

3’

3’

A.nidulans Fig. 4. Possible

secondary

structure

and A. ~i~zll~~s 235 I-RNA sequences

of‘the Ll -associated

region ofE. coii 23s rRN.4 and llo~~ol~)~ous regions of the tobacco

chloroplast

t RNAMet f

pcG

CG GGGUGGAGCAGCCUGGD

1111111,111 I,,, I. ,GCCCACCUC$UCGGACCGG, 205a .-.I/ J 235 rRNA

E.coli

Dahlberg,

AG”

J.E., Kintner,

Sci. USA 75 (1978) ‘E’J

plast

5s

tobacco chloroplasts 3’

”

Edwards,

,\

Nucleotide

ribonucleic

B. and

Cedergren,

tRNAs.

K., Bedbrook,

FEBS

of

of chloro-

in flowering

plants.

R.J.:

The

preliminary vvith

Lett. 63 (1976) 287-290.

J., Dyer, T. and Kossel, H.: 4.5s rRNA

from Zea mczrs chloroplasts

5’

sequence

acid

nidu/un.r compared oftRNA, Met from A~XICV.F~~S

other initiator

GAGG GUC$UC&GGG ’’’’’’’ GA 2010 235 rRNA

binding

co/i. Proc. Natl. Acad.

J. I83 (1979) 595-604.

sequence

GGDAG”

CM.:

ribosomal

Ecarot-Charrier, t RNAye’

E.: Specific

1071-1075.

Dyer, T.A. and Bowman, Biochem.

pcGCGG~~~~~~~~~~~U

C. and Lund,

to 23s rRNA of Escherichia

tRNA,“”

the 3’ end ofprokaryotic

shows structural 23s rRNA.

homology

Biochem.

with

Int. 2 (1981)

533-538. Hori. tRNAye’

DAG”

pCGCGGGG~AG~G~A~~UGG

,GAG 3 8’ ,980

AGACH:GUCGCGG~,

Machatt.

,

rRNA Fig. 5. Sequences tRNA,“”

involved in formation

and 23s rRNAs

of the complex

of E. coli, tobacco

between

chloroplasts

and

A. tlidulrm.

23s

Branlant,

C.: The 33terminal

ribosomal

the 3’-terminal

and with chloroplast

RNA:

region

4.5s rRNA.

Structure

and

of eukaryotic

28s

Nucl. Acids Res. 9

(1981) 1533-1549. Maxam.

.4.M. and Gilbert,

W.: A new method

DNA. Proc. Natl. Acad. Takaiwa,

F. and Sugiura.

and 5s ribosomal

M.: Nucleotide

sequence

from tobacco

of the 4.5s chloroplasts.

180 (1980) l-4.

F. and Sugiura.

in tobacco

for sequencing

Sci. USA 74 (1977) 560-564.

RNA genes

Mol. Gen. Genet. Takaiwa, ACKNOWLEDGEMENTS

in 5S rRNA

tree of 54 5S RNA

Sci. USA 76 (1979) 381-385.

Ebel, J.P. and

with

change

and a phylogenic

of bacterial

homology

rRNA

S.: Evolutionary

Proc. Natl. Acad. M.A.,

region

5’ 235

Osawa, structure

species.

II III II

A.nidulans

H. and

secondary

M.: Heterogeneity

chloroplasts.

Mol.

of 5s RNA species

Gen.

Genet.

181 (1981)

385-389.

We thank Dr. K. Shinozaki for valuable suggestions. The initial part of this work was carried out at the National Institute of Genetics (Japan). This work was supported in part by Grants-in-Aid from the Ministry (Japan).

of Education,

Science

and

Culture

Takaiwa.

F. and

16s.23s chloroplast Takaiwa,

Sugiura.

rRNA

Biochem.

124 (1982b)

Gene

Bram,

R.J.. Young,

site flanking

R.A. and Steitz, J.A.: The ribonuclease

23s sequences

in the 30s ribosomal

III

precursor

RNA of E. co/i. Cell I9 (1980) 393-401. Branlant.

C., Krol, A., Machatt,

structure

of the protein

RNA. Homologies Lll

mRNA

Nucl. Acids Brosius,

with putative

and of a region

structures

of mitochondrial

23s of the

16s rRNA.

Res. 9 (1981) 293-307.

J., Dull. T.J., Sleeter,

organization

region of ribosomal

secondary

and

primary

D.D. structure

tobacco

sequence

chloroplasts.

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Sugiura,

M.: Sequence

for the rRNA

genes

of a

of tobacco

M.: The complete

nucleotide

RNA gene from tobacco

sequence

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17 (1982) 213-218. N., Shinozaki,

K. and Sugiura,

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cloning

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359-363.

A. and Ebel, J.P.: The secondary

Ll binding

2665-2676.

nuclcotide

DNA. Nucl. Acids Res. 9 (1981) 5399-5406.

N. and Sugiura,

Tomioka,

of the

13-19.

region

of a 16s ribosomal REFERENCES

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chloroplast

M.: The complete

gene

N., Shinozaki.

putative Tohdoh.

sequence

DNA. Nucl. Acids Res. IO (1982a)

F. and Sugiura,

of a 23s Tohdoh.

M.: Nucleotide

spacer region in an rRNA gene cluster from tobacco

Tomioka.

N. and Sugiura,

of a 16s

ribosomal

Amcysris rziduhs. Wildernan.

H.F.:

of a ribosomal

Gene

11896-l

1900.

Communicated

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