Nucleotide sequence and transcript analysis of three photosystem II genes from the cyanobacterium Synechococcus sp. PCC7942

85

Gene, 67 (1988) 8.5-96 Elsevier GEN 02439

Nucleotide sequence and transcript analysis of three photosystem II genes from the cyanohacterium Synechococcus sp. PCC7942 (Recombinant

DNA;

p&l);

psbC; Anacystis nidzdans R2; prokaryotic

gene family; operon;

translational

start

codon)

Susan S. Golden and George W. Stearns* Department of Biology, Texas A & M University, College Station, TX 77843-3258 (U.S.A.) Received

15 January

1988

Revised

29 February

Accepted

3 March

1988

Received

by publisher

1988 25 March

1988

SUMMARY

The genome of the cyanobacterium Synechococcus sp. PCC7942 contains two genes encoding the D2 polypeptide of photosystem II (PSII), which are designated here as psbDZ and psbDII. The psbDI gene, like the psbD gene of plant chloroplasts, is cotranscribed with and overlaps the open reading frame of the psbC gene, encoding the PSI1 protein CP43. The psbDII gene is not linked to psbC, and appears to be transcribed as a monocistronic message. The two psbD genes encode identical polypeptides of 352 amino acids, which are 86% conserved with the D2 polypeptide of spinach. In plants, the translational start codon of the psbC gene has been reported to be an ATG codon 50 bp upstream from the end of the psbD gene. This triplet is not present in the psbDI sequence of Synechococcus sp., but is replaced by ACG, a codon which is very unlikely to initiate translation. Translation of the psbC gene may begin at a GTG codon which overlaps the psbDI open reading frame by 14 bp and is preceded by a block of homology to the 3’ end of the 16s ribosomal RNA, a potential ribosome-binding site. There are only two bp differences between the sequences of the two psbD genes; one of these results in substitution in psbDII of GCG for the presumed GTG start codon in psbDI.

The major membrane protein complexes of photosynthetic thylakoids from the oxygenic cyanobacteria are very similar to those from plant

chloroplasts (Ho and Krogmann, 1982). All of the core polypeptides of PSI1 are conserved between these groups of organisms. These include two chlorophyll-a-binding polypeptides, CP47 and CP43, two proteins, D 1 and D2, which may house all of the

Correspondence

methionine;

INTRODUCTION

Abbreviations: Texas

A & M University,

(U.S.A.) * Present Oaks,

to: Dr. S.S. Golden, College

Department

of Biology,

Station,

TX 77843-3258

1900 Oak Terrace

Lane, Thousand

Tel. (409) 845-9824. address:

AMGen,

CA 91320 (U.S.A.)

Tel. (805)499-5725

Ext. 340.

open

reading

acid;

PSII,

rRNA,

aa, amino acid(s); bp, base pair(s); kb, kilobase frame;

sodium dodecyl 0.001 M EDTA,

0378-l 119/8X/$03.50 0 1988 Elsevier

Science Publishers

B.V. (Biomedical

Division)

PIPES,

photosystem

ribosomal

reaction

S, sedimentation

center

of PSII;

constant;

SSPE, 0.1 M NaCl, 0.01 M NaH,

pH 7.6; tRNA,

ORF,

1,4-piperazine-diethanesulfonic

II; P680,

RNA;

sulfate;

fmet, formyl

or 1000 bp; nt, nucleotide(s);

transfer

RNA.

SDS, PO,,

86

cofactors

of the reaction center, and the two subunits

of cytochrome b-559 (reviewed by Satoh, 1985). In higher plants each of these proteins is encoded by a unique

gene in the chloroplast

cyanobacteria

genome,

whereas

one p&4 and psbD gene, respectively (Curtis Haselkorn, 1984; Golden et al., 1986; Williams Chishohn, heterodimer

in

D 1 and D2 are encoded by more than and and

1987). Dl and D2 are thought to form a which binds

the P680 reaction

center

chlorophyll molecule, the primary electron acceptor, and the first and second stable electron accepting quinones,

QA and

Qn .This model was first proposed

by Trebst (1986) based on homology of these polypeptides to the crystallographically-defined L and M subunits of the reaction centers of purple bacteria (Hearst and Sauer, 1984; Deisenhofer et al., 1985). This idea was supported when Nanba and Satoh (1987) isolated PSI1 particles containing only Dl, D2, and cytochrome b-559 which exhibited photoaccumulation of reduced pheophytin. The cyanobacterium Synechococcus sp. PCC7942 (also called Anacystis nidzdans R2) contains three distinct, transcriptionally active psbA genes encoding two forms of the Dl protein (Golden et al., 1986), suggesting that thylakoids from this organism may contain a mixed population of Dl-D2 heterodimers. We report here the nt sequence and transcript analysis of the two DZencoding and the single CP43-encoding ORFs from this organism.

MATERIALS

AND METHODS

(a) Bacterial straius The cyanobacterium Synechococcus sp. (Pasteur Culture Collection #7942) was grown in liquid BG-11 medium (Allen, 1968) with constant shaking at 30’ C under fluorescent illumination at a photosynthetic photon flux density of 100 PEinsteins x S - ’ x rnp2. Alternatively, cells were plated on BG11 solidified with 1.5 % Difco Bacto-Agar and supplemented with 1 mM Na,S,O, as previously described (Golden et al., 1987). A detailed protocol for transformation and gene inactivation in this organism is described by Golden et al. (1987). The psbDII-truncated mutant R2S2.2 was grown in the presence of 40 pg spectinomycin/ml.

(b) Screening of the bacteriophage Southern analysis

library

The psbD and psbC genes were identified library of Synechococcus sp. DNA cloned bacteriophage 2 vector Charon 30 (Rimm 1980). This library L. Sherman.

from a in the et al.,

was a gift from C. Vann

Plasmids

containing

portions

and

and

of the

psbD gene from Chlamydomonas reinhardtii and the psbC gene from Synechocystis sp. PCC6803

were a

gift from J. Williams. Plaques were lifted from agar plates onto Colony/Plaque Screen filters (NEN Research Products), probed with a fragment of the C. reinhardtiipsbD gene which had been 32P-labeled by nick translation, and washed in 2 x SSPE, 0.2% SDS at 65°C (Maniatis et al., 1982). Southern analysis of phage DNA to identify the psbC gene was performed by alkaline capillary transfer of DNA from 0.7% agarose gels to Gene Screen Plus (Reed and Mann, 1985). Unhybridized nick-translated psbC probe sequences were washed from the blot as for the Plaque Screen filters. (c) Nucleotide sequence analysis Restriction fragments of the psbD and psbcgenes were cloned into Ml3 mp 18 and 19 (Norrander et al., 1983), or the Bluescript (Stratagene) and pUCl8/19 (Norrander et al., 1983) plasmid vectors for nt sequence analysis. Escherichia coli strain DH5a (BRL) was used as a host for all plasmids, and the Ml3 vectors were propagated in JMlOl (Messing, 1983). A combination of dideoxynucleotide (from single- or double-stranded templates) and chemical cleavage methods was used to determine the complete nt sequence of each of the three genes on both DNA strands (Sanger et al., 1977; Chen and Seeburg, 1985; Maxam and Gilbert, 1980). (d) Transcript analysis Total RNA was isolated from Synechococcus sp. cells as described elsewhere (Golden et al., 1987). Northern analysis was performed by capillary transfer of glyoxal-treated RNA samples from agarose gels to Gene Screen Plus membranes as described in the legend to Fig. 4. Blots were probed with 32P-labeled restriction fragments produced by nick translation with a BRL labeling kit or anti-sense

81

RNA transcribed

from Bluescript

mids (Stratagene). identified

using

vector-based

plas-

The 5’ ends of transcripts

were

nuclease-protection

extension methods probes for mapping script are described

and

RESULTS

(a) Identification of the psbD genes from SynechocOccuSsp.

primer-

as described for Fig. 5. The the 5’ end of the psbDII tranhere. For primer extension, a

Recombinant Kharon bacteriophage containing two different EcoRI fragment inserts of Synechococ-

17-mer was synthesized which was complementary to the sequence shown in Fig. 2 at positions -2 to -18 of the psbDII 5’ flanking region. The extended quencing

cus sp. DNA were identified by hybridization with a C. reinhardtiipsbD probe which had been 32P-labeled by nick translation. The inserts of 11.5 kb and 10 kb

product was measured against a seladder generated by dideoxynucleotide

chain termination

AND DISCUSSION

corresponded

using the same primer on a DNA

to two

bands

digests which hybridized

template containing the psbDII 5’ flanking region. The DNA fragment used for nuclease protection experiments was 5’ end-labeled at the HincII site inside the psbDII ORF (nt 146), and extended to a PvuI site 173 bp upstream from the start codon. The same labeled fragment was subjected to chemical cleavage reactions (Maxam and Gilbert, 1980) to generate a sequencing ladder by which to measure the protected fragment.

in genomic

EcoRI

to the probe in Southern

analysis (data not shown). Subsequent restriction mapping and Southern analysis indicated that the region of psbD homology in each of these clones was completely internal to the EcoRI fragments, suggesting that two distinct psbD genes were present in this organism, as has been reported for the cyanobacterium Synechocystis sp. PCC6803 (Williams and Chishohn, 1987). The nt sequence of both regions of homology confirmed the presence of two complete psbD ORFs. These genes were named psbDI and psbDII, in keeping with the nomenclature of the cyanobacterial psbA multigene families (Curtis and Haselkorn, 1984; Golden et al., 1986). Fig. 1. sche-

psbC ORF

Pv 4

HcA

SB

Hc

44

44

4

psbDII ORF

Fig. 1. Schematic

summary

of the psbD physical

each of the psbD genes and psbC. Restriction identify each enzyme:

A, ApaI;

B, BumHI;

H, HindIII;

is represented

by an open box labeled with the respective

The direction,

size, 5’-end position

restriction

fragment.

and estimated

heavy lines represent

sites are denoted

Hc, HincII;

psbDZ and psbDZZ are aligned with respect

representing

I

maps. Horizontal

enzyme cleavage

ioodp

arrows

P, PstI; Pv, PvuII; R, EcoRV;

to conserved gene name;

3’-end position

the region of the chromosome

by downward

restriction

sites and ORFs.

the orientation

of each transcript

abbreviations

S, SstI; Sp, SphI. The heavy lines The position

of each of the ORFs

of each ORF is, left-to-right,

is denoted

which contains

and the following

by a hatched

N to C terminus.

arrow below the respective

88

-170 . RSU GCTTCCAGTA TCTCAGATCA ATATCCCTCC CCGATGGGAG GCAGCCTGCC -GAGA CGACAGTAAC GAAACGTTAA GCTGCGACCT CTGAGAGGGG CAGTTTCTGC GATTTCAATG AGTAGATCTG CTCAAAGGCT TGCCCCCAAA GGTCTCTGAG CAATTCGTGT CGGAm

-80

(-81)

Q&L

CGGTTGCCAT AATCATCCAT GTTTGTCGCA AACTGCGTTT CTCTGAACTG ATATTGCAAA TATCTocAc;A TTGCTAAGCA +l AAATCCTTGG AGCCGAGGGGTGGA CAACTTGCTG AAACGTTCTG ATTTCAGTAC GGCRAGAGOT TTTAGATCCA ATG ACG ATT (9) AAA M T I r31 GCA GTA GGG CGA CXG CCA GCG GAG CGG GGA TGG TTT GAC GTC CTC GAC GAC TGG CTG AAG CGC GAC CGA TTT GTA (84) AVGRAPAERGWFDVLDDWLKRDRF V WI

TTT GTG GGT TGG TCA GGG TTG CTG CTG TTT CCC TGT GCG TAT TTA GCA CTG GGC GGG TGG TTG A& FVGWSGLLLFPCAYLALGGWLTGT TTT GTG ACG TCG TGG TAC ACC CAC GGC ATC GCG TCT TCG TAC TTA GAA GGC ‘XC FVTSWYTHGIASSYLEGGNFLTVA

GGG ACC AGC (159) S

[531

AAC Tl!T TTG ACC GTA GCA GTG (234) V

[781

AGC ACC CCA GCG GAT GCG TTT GGG CAT TCG TTG ATG CTG CTG TGG GGC CCC GAG GCA CAA GGG AAC TTC GTG CGT (309) STPADAFGHSLMLLWGPEAQGNFV R [103] TGG TGC CAG TTG GGT GGC TTG TGG AAC TTC GTA GCA CTG CAC GGC GCC TIC GCG CTG ATT GGG TTC ATG CTG CGT (384) WCQLGGLWNFVALHGAFALIGFML R (1281 CAA TTT GAG ATT GCG CGG TTG GTG GGC GTC CGT CCG TAC AAC GCG ATC GCC TTT TCG GGT CC% ATC GCA GTG TTC (459) QFEIARLVGVRPYNAIAFSGPIAV F [153]

GTG TCG GTG TTC TTG ATG TAC CCG TTG GGT CAA TCG AGC TGG TTC TTC GCT CCG AGC TTT GGC GTG GCA GCG ATT (534) VSVFLMYPLGQSSWFFAPSFGVAA I (1781 TTC CGG !tTT TTG TTG TTC CTG CAA GGG TIC CAC AX FRFLLFLQGFHNWTLNPFHMMGVA

TGG ACC TTG AAC CCA TTC CAC ATG ATG GGC GTG GX

GGG (609) G [203]

ATT TTG GGT GGG GCA TTG CTG TGC GCC ATT CAC GGT GCG ACG GTG GAG AAC ACC CTG TTC GAG GAT TCA GAG CAA (684) ILGGALLCAIHGATVENTLFEDSE Q (2281 TCG AAC ACC TTC CGG GCA TTT GAG CCG ACG CAG GCC GAA GAG ACG TAC TCG ATG GTG ACG GCG AAC CGT TTT TGG (759) SNTFRAFEPTQAEETYSMVTANRF W [253] AGC CAG ATT TTC GGG ATT GCG TTT TCG AAC AAG CGG TGG CTG CAC TTT !M’C ATG CTG TTC GTG CCG GTG ACG GGC (834) SQIFGIAFSNKRWLHFFMLFVPVT G (2781 TTG TGG ATG AGC TCG ATC GGG ATT GTA GGT TTG GCG TTG AAC CTG CGG GCG TAC GAC TPC GTG TCG CAG GAG CTG (909) L (3031 LWMSSIGIVGLALNLRAYDFVSQE CGG GCC GCT GAG GAT CCG GAA TTT GAG ACG TTC TAC ACG AAG AAC ATC TI’G TTG AAC GAA GGG ATT CGG GCC TGG (984) W (3281 RAAEDPEFETFYTKNILLNEGIRA T C ATG GCA CCG CAA GAC CAA CCG CAC GAA AAA TTC GTC TTC CCC GAA GAG GTT CTG CCC CGT GGT AAC GCT CTC TAG (1059) * [352*] MAP QD QPHEKFVFPEEVLPRGNAL acg aaa aat tcg tct tee ccg sag agg ttc tgc ccc GTG GTA ACG CTC TCT AGT(1060) v/M V T L S tknssspk rfcp S (61 (end PsbDl-I homology) CCT TCC GTG ATC GCA GGC GGC CGG GAT ATT GAC TCC ACC GGT TAC GCT TGG TGG TCC GGC AAT GCC CGT TTG ATC (1135) I (311 PSVIAGGRDIDSTGYAWWSGNARL

Fig. 2. Nucleotide

and deduced

region represents

the nucleotide

as + 1 and subsequent

nt numbers

amino acid sequences sequence

are shown in parentheses

-1 to -170. Within the ORF, a single sequence shown

above the line. Representation

ORF overlaps

of the psbDZ, psbDZZ, and psbC genes. The upper line in the upstream

flanking

of the psbDZZ gene and the lower line is psbDZ. The first nt of the psbD ORF is designated represents

at the end of each line of nt sequence;

5’ flanking

sequences

are numbered

both genes, except at nt 1041 and 1044, where the psbDZZ nt differences

of psbDZZ ends after nt 1059; the downstream

the end of the psbDZ ORF. The ACG that corresponds

for psbC is shown in lower case letters, as is the translation

to the position

flanking

are

region can be seen in Fig. 3. The psbC

of the previously

of this region. The upper case indicates

reported

translational

our suggested

start codon

translational

start

site at nt 1043. Amino acids are specified by the single letter code shown below the nt sequence. Amino acid numbers are given in brackets

89

AAC CTG TCC GGT AAG CTG CTG GGC GCT CAC GTC GCT CAT GCT GGC TTG ATC GTC TTC TGG GCT GGT GCG ATG ACG (1210) NLSGKLLGAHVAHAGLIVFWAGAM T [561 CTG TTT GAA GTC GCG CAC TTT GTC CCC GA?+ AAA CCG ATG TAC GAG CAA GGC ATC ATC CTG CTC TCG CXC TTG GCG (1285) LFEVAHFVPEKPMYEQGIILLSHL A [811 ACC CTC GGC TGG GGC GTT GGC CCT GGT GGC GAA GTC OTC GAT ACC TTC CCC TAC TTT GTG G'IT GGG GTT CTG CAC (1360) TLGWGVGPGGEVVDTFPYFVVGVL H [106] CTC ATT TCT TCC GCC GTT CTG GGT TTG GGT GGG ATC TAC CAC GCC CTG CGC GGC CCT GAG TCG CTG GAA GAG TAC (1435) LISSAVLGLGGIYHALRGPESLEE Y [131] ACX ACC TTC TTC AGC CAA GAC TGG AAA GAC AAG AAT CAG ATG ACC AAC ATC ATT GGT TAT CAC CTG ATT CTG CTG (1510) STFFSQDWKDKNQMTNIIGYHLIL L [156] GGC TTA GGT GCC TTC TTG CTG GTC TTT AAG GCC ATG TTC TTC GGC GGT GTC TAT GAC ACC TGG GCG CCG GGT GGT (1585) GLGAFLLVFKAMFFGGVYDTWAPG G [181] GGC GAT GTC CGC ATC ATC TCC AAC CCA ACC CTC AAC CCG GCT GTG ATC 'iTC GGC TAC CTG CTG AAA TCA CCC TTT (1660) GDVRIISNPTLNPAVIFGYLLKSP F [206] GGT GGC GAC GGC TGG ATT GTC AGC GTC GAC AAC CTT GAA GAC GTG ATT GGC GGC CAT ATC TGG ATT GGT CTG ATC (1735) GGDGWIVSVDNLEDVIGGHIWIGL I [231] TGC ATT TCG GGT GGT ATC TGG CAC ATC CXG ACC AAG CCT TTT GGC TGG GTC GGT CGC GCC TTC ATC TGG AAT GGC (1810) CISGGIWHILTKPFGWVGRAFIWN G [256] GAA GCT TAC CTC TCC TAC AGC TTG GGT GCC CTG TCG TTG ATG GGC T'PC ATT GCC TCG ACG ATG GTT TGG TAC AAC (1885) EAYLSYSLGALSLMGFIASTMVWY N [281] HindIII AAC ACC GTC TAT CCT TCC GAG TTC TTT GGC CCG ACC GCT GCT GAA GCT TCG CAA TCG CAA GCC TTC ACC TTC TTG (1960) NTVYPSEFFGPTAAEASQSQAFTF L [306] GTG CGT GAC CAA CGC CTC GOP GCC AAC ATC GGT TCA GCT CAA GGC CCG ACC GGT CTG GGT AAA TAC CTG ATG CGC (2035) VRDQRLGANIGSAQGPTGLGKYLM R [331] TCT CCT ACC GGC GAG ATC ATfZ TTC GGT GGC GAA ACC ATG CGC TTC TGG GAC TTC CGT GGC CCJ! TGC GTG GAG CCC (2110) SPTGEIIFGGETMRFWDFRGPCVE P [356] CTG CGT GGA CCG AAT GGT CTG GAT CTC GAC AAG CTG ACC AAT GAC ATT CAG CCT TGG CAA GCC CGT CGT GCG GCT (2185) LRGPNGLDLDKLTNDIQPWQARRA A [381] GAG TAC ATG ACC CAC GCA CCG CTG GGT TCG CTG AAC TCT GTG GGT GGT GTG GCA ACG GAA ATC AX EYMTHAPLGSLNSVGGVATEINSV

TCG GTG AAC (2260) N [406]

TTC GTG TCT CCC CGT GCT TGG TTG GCG ACC AGC CCA TJ?C GTC TTG GCC TPC TTC TTC TTG GTC GGT CAC CTC TGG (2335) FVSPRAWLATSPFVLAFFFLVGHL w [431] k&L CAT GCA GGC CGC GCT CGT GCA GCT GCT GCA GGC TTT GAG AAA GGT ATC GAT CGC GCG ACC GAA CCC GTG CTC GCA (2410) HAGRARAAAAGFEKGIDRATEPVL A [456] ATG AGA GAC CTC GAC TAA TTCCA?iCTGC AGGACATTAG CCTCAAAGIC TGAAAAGCCC TTGCCTCGGC AGGCGGTTTT TCGTATCTCT(2498) [461*] MRDLD* GGGGGAATGA CAACGCCTGC GAGCAGCTGG GGTGCTCTTA CCGACAGTGG TTTGGGAGAA GTCACTGAGC GGCTCTAGTT TTCTGGAATC

(2588)

TTGCGGTGGA ATAGTCCCAG

(2678)

CTCGCAGCCC TCTCGTCGCA AAGTCTCAGT TAGACGCTGC CAGTTCGCAG CATCGCAAG?i GCATTTCTCC

at the end of each line; numbering Possible

ribosome-binding

are marked

by arrowheads

of prokaryotic underline.

ofthe psbD aa sequence

the 5’ end (start points)

(one for the upper line and three for the lower line). Blocks of nt having sequence

‘-10’ elements

are underlined.

No such element was identifiable

the nt sequence;

begins at the M (ATG, nt l-3) and of psbC at the V/M (GTG, nt 1043-1045).

sites are shown in bold face type. Those nt that represent The ‘-35’ element upstream

upstream

from the psbDZ/psbC message

from psbDZ2. Only those restriction

there are other sites for some of these enzymes

that are not shown.

sites mentioned

of each of the messages

and spacing characteristics is marked

with a zig-zag

in the text are shown above

90

psbDI

I+1040 CCC CGTGGT

t '1060 AAC GCT CTC TAG TCCTTCCGTG

psbDII D2

CCT CGCGGT PRGNAL*

AAC

psbDI psbDII

’ 1090 TGACTCCACC TCAACGGCCT

I

GCT CTC TAG GCATTTTTCT

I

GGTTACGCTT GGTGGTCCGG CCCCAAAGGA GTCTTGCCGT

I

I

ATCGCAGGCG CTAACAGGAA

GCCGGGATAT AGATTTTTGC

I

I

,

CAATGCCCGT TAGATAGGGG

TTGATCAACC GCGTAAACCT

TGTCCGGTAA GTTGTAGTTG

Fig. 3. Divergence of the psbD genes downstream of the ORF. The nt sequences encoding the last six aa of the D2 C-terminus and downstream flanking region are compared for psbDI and psbDIZ. Numbers refer to the numbering system used for Fig. 2. Two nt that differ between the ORFs ofthe two genes are shown in bold type. There is no apparent conservation between the two sequences following the TAG stop codons.

matically

summarizes

the physical map of the genes

as derived from restriction and transcript analyses. (b) Nucleotide

mapping,

nt sequence,

sequence analysis of the psbD genes

The psbDZ and psbDZZ genes have ORFs encoding 352 aa (Fig. 2). Only two bp differences were detected within the coding regions of the two genes; both of these occur in third positions of codons, and do not affect the specified aa. The overall conservation between the aa sequence of this D2 polypeptide and that reported for spinach is 86%, although an additional aa is encoded within the N-terminus of the spinach psbD gene (Holschuh et al., 1984). Since the two genes predict an identical D2 polypeptide, and since the three psbA genes encode two distinct forms of the Dl polypeptide (Golden et al., 1986), there are two possible Dl-D2 heterodimers in the PSI1 reaction centers. The flanking regions downstream from the ORFs diverge immediately following the stop codons (Fig. 3). This is not surprising, since psbDZ overlaps a second ORF, and no equivalent ORF is present adjacent to psbDZZ (see RESULTS AND DISCUSSION, section c). Except for the 2 nt immediately before the start codons, the upstream regions of psbDZ and psbDZZ are divergent. The start codons of both genes are preceded by a Shine and Dalgarno (1974) consensus site which is complementary to the 3’ end of one of the Synechococcus sp. 16s rRNA genes (S.S.G., unpublished). The spacing between the putative ribosome-binding sites and the ATG start codons is 10 bp for psbDZ and 11 bp for psbDZZ. This is within the range of spacing observed in E. coli; however, the usual distance between these elements in E. coli is 7 + 2 nt (Stormo, 1986; Gold, 1988).

(c) Identification psbC gene

and nucleotide

sequence

of the

Previous reports indicated that the psbC gene in plants and in another cyanobacterium overlaps the 3’ end of the psbD gene, potentially by 50 bp (Holschuh et al., 1984; Bookjans et al., 1986; Williams and Chishohn, 1987). Examination of the nt sequences of the C-terminal regions of the two psbD genes did not reveal the ATG putative start codon for psbC gene that is present in the other organisms. Southern analysis of DNA from each of the Kharon bacteriophage clones indicated that homology to psbC lay downstream from the psbDZ gene, but no psbC homology was present adjacent to psbDZZ (data not shown). The sequence of the DNA fragment downstream from psbDZ confirmed an ORF that was highly homologous to the spinach psbC sequence, extending from nt 1007 to nt 2425 (Fig. 2); however, the putative ATG start codon was replaced by the triplet ACG in the Synechococcus sp. sequence. Since other PSI1 genes from this organism appear to be lethal in E. coli when cloned on highcopy-number plasmids (Golden et al., 1986), we wanted to determine whether a T + C transition had occurred during subcloning. Therefore we determined the sequence at this position by chemical cleavage directly from the recombinant bacteriophage DNA, and found that ACG was present in the original clone. A report by J. Gingrich (Workshop on Molecular Biology of Cyanobacteria, July 1987, St. Louis, MO) that the marine cyanobacterium Synechococcus sp. PCC7002 also lacked the ATG triplet supports our sequence. Gingrich suggested that translation from the psbC transcript might begin at a conserved GTG codon nearer the end of psbD in chloroplasts and in cyanobacteria (Table I). GTG

91

TABLE I Nucleotide sequence a comparison of the psbD/psbC overlap region from four species GACTCAAGATCAGCCTC~AAAACCTTATATTCCCCTGAGGAGGTTCTACCAC~G~GGCTCAAGATCAGCCTC~AAAACCTTATATTCCCTGAGGAGGTTCTACCCC~GAATCCCCAAGATCAACCCC~AAAACTTTATCTTTATCTTCCCTGAGGA~TTCTCCCCC~GTAACCGCAAGACCAACCGCACGAAAAATTCGTCTTCCCCGAAGA~TTCTGCCCC~GTA-

Pea” Spinach” Synechocystisd Synechococcus’

a Corresponds to the region represented as nt 990-1048 in Fig. 2. Bold type indicates possible ribosome-binding sites; potential start codons are underlined. b Bookjans et al. (1986). ’ Holschuh et al. (1984). d Chisholm and Williams (1988). e Fig. 2, nt 990-1048.

initiates translation in 8 y0 of the known E. coli genes (reviewed by Stormo, 1986), and fmet-tRNA will form a ternary complex with a 30s ribosomal subunit and either a GTG or ATG start codon in vitro (reviewed by Gold et al., 1981). At least one cyanobacterial gene is thought to initiate translation at a GTG (Lomax et al., 1987). Overall aa homology among proteins specified by the psbC genes of Synechococcus sp. and spinach is 79-80x, depending on whether the comparison is made beginning at nt 1007 or 1043, respectively. A number of features in the sequence (Fig. 2) support Gingrich’s hypothesis. Preceding the GTG codon (nt 1043-1045), with a 9-bp spacer, is the sequence ‘AAGAGG’, a perfect complement to the 3’ end of one of the 16s rRNA genes (S.S.G., unpublished results). The best match to a putative ribosome-binding site upstream from the ACG codon is ‘AAGA’ (with an 8-nt spacer). The sequences from spinach (Holschuh et al., 1984), pea (Bookjans et al., 1986), Synechocystis sp. (Chisholm and Williams, 1988), wheat (J. Gray, personal communication) and barley (E. Neumann, personal communication), all of which have an ATG at the site previously reported as the psbC start codon, have a higher degree of similarity to the Shine-Dalgamo sequence upstream from the GTG codon than upstream from the ATG (Table I). It should be noted, however, that the degree of homology to the 16s ribosomal RNA is not sufficient to predict the strength of ribosome binding (Gold et al., 1981). There are only two bp differences between the psbDI and psbDII sequences, neither of which changes an aa in the D2 polypeptide. However, these two nt differences would alter the aa predictions for an overlapping psbC gene in another reading frame.

One difference changes the putative GTG psbC start codon in psbDI to a GCG triplet in psbDII. One might expect the psbC start codon to be missing from psbDII since there is no psbC ORF on its message. This nt change may block ribosomes from abortive translation initiation at the end of psbDII. The ACG triplet, however, is present in both psbDI and psbDII. It is unlikely that the Synechococcus sp. translational start site differs from that used by Synechocystis sp. and chloroplasts. The similarity of the genes between species, including the positioning of Shine-Dalgarno sequences before each of the potential start codons, suggests that the initiation signals are conserved. Therefore these data suggest that GTG is the start codon for psbC in all of these species. Protein sequence analysis of the mature CP43 polypeptide from spinach (Michel et al., 1988) does not clarity the translational start site, as there is evidence of protein processing during maturation. The mature N-terminal residue is N-acetyl-O-phosphothreonine, which represents the third aa if translation begins at the conserved GTG, and the fifteenth residue if translation begins at the first ATG in the spinach sequence. We have not yet determined whether the CP43 polypeptide or three other PSI1 phosphoproteins are modified in the same way in cyanobacteria. (d) Analysis of transcripts from the psbD genes Northern analysis of total RNA probed with a restriction fragment containing most of the psbDI ORF detected a 2.5-kb RNA (Fig. 4, lanes 1 and 2). A probe from a region of the psbC gene that did not overlap psbDI recognized an RNA of the same size

92

12

34

upstream

56

from psbDI would be expected

nate shortly psbC. No mRNA

bands

were detected

using

highly

homologous

to termi-

stop codon

of

other than the 2.5-kb species a probe to

that

a message

should

be very

arising

from

psbDII. To determine whether a psbDII transcript was present, we constructed a sensitive probe that

2.5kb

d== LZkb LO kb

Fig. 4. Northern truncated glyoxal

mutant and

according

of psbDI, psbC, and psbDII. Total

transfers

cells (lanes 1, 3, and 5) and the psbDZZ-

RNA from wild-type

R2S2.2

separated

to Thomas

(lanes 2, 4, and 6) was treated

by

using electrophoresis hybridized BumHI

1.2%

with

gel electrophoresis,

nylon membrane

from the

by capillary

transfer

buffer. The blot was sliced into strips and

with the following fragment,

agarose

(1983). The RNA was transferred

gel to a positively-charged

probes:

32P-labeled

lanes 1 and 2, a 1.2-kb

by nick translation,

ending

at

nt 921 (Fig. 2) and carrying

most of psbDZ and none of psbC;

lanes 3 and 4, and antisense

RNA transcribed

consisting

of a 435-bp HindIII-PstI

from a template

fragment

from a template

immediately to -158).

upstream Unhybridized

consisting probe

was washed

0.1 x SSPE, 0.2% SDS at 70°C (Maniatis mark the hybridizing dots indicate

transcripts,

the positions

leading edges of the rRNAs

RNA trans-

of a 154-bp D&I

the psbDZI ORF

from

to psbC

internal

(Fig. 2, nt 1930 to 2365); lanes 5 and 6, an antisense cribed

after the translational

fragment

(Fig. 2, nt -4

from the blots in et al., 1982). Arrows

whose sizes are shown. Black

of the rRNAs; are artifacts

diffuse bands caused

at the

by these abun-

dant RNA species.

(Fig. 4, lanes 3 and 4). This result suggests that the 2.5-kb species is the transcript from an operon that includes psbDI and psbC. To determine whether psbDI and psbC are cotranscribed, the 5’ end of the psbDI message was mapped using Sl and mung bean nuclease-protection and primer-extension reactions (Fig. 5). These analyses identified a single start point with terminal nt corresponding to nt -53 to -51 in Fig. 2. This 5’ end was very abundant in the RNA population, indicating that it corresponded to the message detected by Northern analysis. A 2.5-kb message that begins approximately 50 nt

would detect a rare psbDII message, but would not hybridize to the psbDI message. This probe was transcribed as a 32P-labeled antisense RNA from a DdeI fragment (Fig. 2, nt -4 to -158) that would recognize the 5’-untranslated leader sequence, but none of the protein-coding region, of a psbDII message. A Northern blot probed with this labeled RNA identified an approx. 1.2-kb message from psbDII (Fig. 4, lane 5). This species was not detected by any nick-translated probes we used, including the DdeI fragment, indicating that the psbDII message is much less abundant than the psbDI message in cells grown under our standard laboratory conditions. Sl nuclease mapping of the psbDII transcript using a probe that originated at a &XII site inside the psbDII ORF (Fig. 2, nt 146) and extended to nt -173 identified a cluster of protected bands approx. 100 nt upstream from the ORF (data not shown). This Sl-resistant signal had less than onetenth the intensity of a protected band extending a few nt upstream from the start codon. The more intense signal probably originates from protection of the psbDII-derived DNA fragment by the abundant psbDI message, which is complementary to psbDII within the ORF and 2 nt upstream from the start codon (Fig. 2). The 5’ end of the psbDII transcript was determined more precisely by primer extension using a synthetic oligodeoxynucleotide that was complementary to the 5’-untranslated region of the psbDII message (nt -2 to -18). To assign the position of the extended product, the same primer was used for dideoxynucleotide sequencing from a DNA template of the psbDII upstream region and the products of this reaction were run as a standard (data not shown). Reverse transcription with this primer yielded a single band corresponding to the nt at -108 indicated in Fig. 2. The sequences upstream from the 5’ end of each message were examined for putative promoter sequences centered around 10 and 35 nt from the

93

transcriptional start points. The psbDI gene has appropriately spaced regions of homology to the ‘-10’ and ‘-35’ elements of E. coli promoters. The psbDII transcript is preceded by a perfect E. coli consensus ‘-10’ element, but no homology to a ‘-35’ element is observed. It should be noted that in E. co& genes under positive regulation often lack homology to the ‘-35’ element (reviewed by Raibaud and Schwartz, 1984). There is insufficient data to define a consensus promoter in Synechococcus sp. or in any other cyanobacterium; however, all five Synechococcus sp. PSI1 genes for which transcripts have been examined are preceded by at least one of the characteristic E. coli promoter elements at the appropriate distance from the transcriptional start point (Golden et al., 1986). Thus, some characteristics of transcriptional initiation signals seem to be conserved between Synechococcus sp. and E. coli. (e) Truncation of the psbDII

gene

A mutant having a truncated psbDII gene was constructed by recombination of a cloned, altered allele with the chromosome of Synechococcus sp. To same end-labeled to generate

fragment,

ORF (numbering as described protection

followed

and Geiduschek

the procedure

for Sl protection

of MBN buffer

with ethanol

and nuclease

ated on 6% polyacrylamide, (Maxam

are shown:

C + T sequencing

message.

Total RNA from wild-type

protection contained labeled

and

of the psbDZ

point)

cells was used for nuclease-

primer-extension

analyses.

Each

reaction

10 pg of total RNA and approx. 21000 cpm of 5’-endDNA.

was a 440-bp

For nuclease

protection

double-stranded

only one 5’ end, corresponding

experiments,

restriction

fragment

to the HincII

to the upstream

(see Fig. 1). The reverse-transcription

primer

the complement

labeled

difference

BumHI

consisted

the chemically

and the nuclease-treated

site

heavy band primer.

of the

ladder;

of the ladder

to the bands

cleaved

by an arrow

in

shown is

in Fig. 2, and that a 1.5~nt migration sequencing

and reverse-transcribed

in lane 3 marked

lane 5, Sl nu-

sequence

presented

lane 2, reaction;

Note that the sequence

of the sequence

which

The following

reaction;

of the 5’ ends took into account

the

by chemical

lane 3, primer-extension

by asterisks.

between

gels (Maniatis

experiments.

The nucleotide

of

were separ-

1980) of the same fragment

lane 1, A + G sequencing

reaction.

lanes 3-5 marked assignment

at

reactions

ladders generated

protection

ladder;

1 mM

and dried. The products

is given to the left, with the nt corresponding

the DNA

site within

psbDZ ORF (nt 146), and extending

clease-protection

50 mM NaCl,

protection

lane 4, mung bean nuclease-protection of the 5’ end (start

was done

for 1 h, after which the

7 M urea sequencing

and Gilbert,

was used in the nuclease

Fig. 5. Determination

et al. (1983). The reaction

Mung bean nuclease

acetate,

at 37°C continued

et al., 1982) along with sequencing

samples

of Turner

was added to the sample in a 300 pl’aliquot

sample was precipitated

cleavage

was performed

(1982). Sl nuclease

experiments.

(30 mM sodium

Incubation

primer extension

with Hue&

at nt + 22 of the

step of the mung bean nuclease

(4 units, Pharmacia) ZnCl,).

by digestion

that terminated

as in Fig. 2). Primer extension

by Kassavetis

hybridization as described

shortened

a 125-bp fragment

ladders

products.

The

is the unextended

94

construct this mutant, a plasmid clone carrying most of the psbDII gene, and unable to replicate in

mechanism

Synechococcus sp., was cleaved at a unique SstI site within the psbDII ORF and ligated to the B fragment from pHP4552 (Prentki and Krisch, 1984). The

psbC. We have not detected a monocistronic message for psbC; however, our analyses may not have been sufficiently sensitive to detect one which

52fragment encodes resistance to spectinomycin and streptomycin and is flanked by inverted repeats that

might be present at a much lower abundance dicistronic message. As the Northern

carry signals.

transcription

and

Transformation

translation

termination

of Synechococcus with this plasmid

sp. to

spectinomycin

resistance

resulted

in replacement

of the psbDII gene with the mutant

change the ratio of D2 : CP43, which might favor a to uncouple

transcription

shown in Fig. 4 demonstrate, was detectable

than the analyses

the psbDII message

only when an antisense

specific for the transcript

of psbD and

RNA probe

from psbDII was used.

allele and loss of the non-replicating vector sequences, as has been previously reported for gene

(f) Conclusions

inactivation in this organism (Golden et al., 1986; Golden et al., 1987). Fig. 4 lane 6 shows that the psbDII message from this mutant is truncated by approximately 200 nt, whereas the psbDI/C message is unperturbed (lanes 2 and 4). The location of the spectinomycin insertion is 213 bp from the end of the ORF; the size of the mutant message suggests that termination is occurring near the junction of psbDII and the spectinomycin-resistance cassette. Viability of the strain carrying a truncated psbDII gene suggests that either psbDII is not essential, or that the mutation did not adversely affect the protein. Given that the mutation removes 71 aa from the C terminus, including all of the hydrophilic lumenal tail and a portion of a putative membrane-spanning domain (Trebst, 1986), it is unlikely that the defective protein would integrate into the membrane and function properly. An indication that psbDII is not essential under normal growth conditions is that a second mutant, carrying a kanamycin-resistance

(1) The genome of the cyanobacterium Synechococcus sp. PCC7942 (A. nidulans R2) contains two psbD genes that encode the PSI1 polypeptide D2. This organism also has been shown to have three functional psbA genes encoding the D 1 polypeptide. Dl is another PSI1 protein that probably forms a heterodimer with D2 to house the cofactors that carry out the primary photochemical reactions. Unlike the psbA genes, which encode two distinct primary aa sequences for Dl, the two psbD genes encode identical D2 proteins. This indicates that only two forms of the PSI1 reaction center are possible in this organism: those having heterodimers consisting of a single D2 polypeptide and one of two forms of the Dl protein. (2) One of the psbD genes, psbDI, is transcribed as a dicistronic message with the unique psbC gene, whose ORF appears to overlap that of psbDI. This is the same configuration that has been reported in higher plant chloroplast genomes (Holschuh et al.,

cassette inserted into psbDI1 after only 93 aa of the ORF, is also viable (data not shown). The presence of a duplication of the psbD locus (but not of psbC) in cyanobacteria presents an evolutionary puzzle. Chloroplast genomes have a psbDC operon analogous to the psbDI configuration, but no examples are known for a second copy of psbD in plants. In the case of the multiple psbA genes in Synechococcus sp. two distinct forms of the D 1 protein are produced, which may suggest a regulatory role if the two products have different functions. The products of the two psbD genes are identical, so any regulatory advantage in maintaining two independently transcribed genes is likely to be of a quantitative, rather than a qualitative nature. It may be advantageous under some conditions to

1984; Bookjans et al., 1986) and in the distantly related cyanobacterium Synechocystis sp. PCC6803 (Chisholm and Williams, 1988). The psbDII gene appears to be transcribed as a monocistronic message. The steady-state level of this message is less than that of the psbDI/Ctranscript by at least lo-fold under our standard growth conditions. (3) Both psbD genes have upstream sequence elements that are similar to transcription and translation initiation signals in E. coli. (4) The psbC gene of cyanobacteria and chloroplasts appears to begin with a GTG codon 14 nt before the psbD stop codon, rather than at a triplet overlapping psbD by 50 nt as previously reported for chloroplast sequences.

95

plast thylakoid

ACKNOWLEDGEMENTS

binding

We gratefully acknowledge the expert technical assistance of M. Nalty, and the contributions of Drs. D.-P. Ma and R. VonderHaar, past and present directors of the Department of Biology, DNA Sequencing Support Facility. We are indebted to Dr. J. Williams, who made sequences and probes available to us prior to publication. This work was supported by a grant from the NIH to S.S.G. (ROl GMS 37040). Some of the equipment used in this research was provided by an NSF Biological Instrumentation Program Grant to S.S.G. and other investigators (BBS-8703784).

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