The Nicotiana chloroplast genome IX. Identification of regions active as prokaryotic promoters in Escherichia coli

Gene, 31 (1984) 23-30

23

Elsevier GENE

1086

The Nicotiana chloroplast genome IX. Identification of regions active as prokaryotic promoters in Escherichia coli (Recombinant DNA; plasmid vector pKO1; ribosomal RNA; tRNA)

X.F. Kong, P.S. Lovett * and S.D. Kung Department of Biological Sciences, University of Maryland Baltimore County, Catonsville, MD 21228 (U.S.A.) 455-2249 (Received

March

(Revision

received

(Accepted

Tel. (301)

6th, 1984) April 27th, 1984)

May 2nd, 1984)

SUMMARY

ThegalK-expression plasmid vector system pK0 1 has been used to clone Nicotiuna chloroplast (ct) promoters that function in Escherichiu coli. The randomly cloned promoter-containing restriction fragments have been located on the ct genome and originate both from those regions encoding ribosomal and transfer RNAs and from locations elsewhere on the ct genome. The results provide the first demonstration that sequences which function as prokaryotic promoters exist in the ct genome.

INTRODUCTION

The ct genomes of many species of Nicotiuna have been mapped with restriction enzymes (Fluhr and Edelman, 198 1; Jurgenson and Bourque, 1980; Tassopulu and Kung, 1983; Seyer et al., 1981), and several ct genes have been cloned in E. coli (Zhu et al., 1982; Sugiura and Kusada, 1979). Expression of the genes for the LS of RuBPCase and some other unidentified polypeptides was demonstrated in both E. coli and Bacillus subtilis (Zhu et al., 1982; 1984). The genes for all ribosomal RNAs, several transfer * To

whom all correspondence

and reprint

requests

should be

directed. Abbreviations: ctDNA,

Ap, ampicillin;

chloroplast

RuBPCase,

ribulose

bp, base pairs;

ct, chloroplast;

DNA; kb, kilobase pairs; LS, large subunit; 1,5-bisphosphate

carboxylase;

rRNA, ribo-

somal RNA.

0378-l 119/84/$03.00

0

1984 Elsevier

Science

Publishers

RNAs and a number of ct proteins have been localized on the genome (Lin and Kung, 1984). Furthermore, several of these genes have been sequenced (Shinozaki and Sugiura, 1982; Takaiwa and Sugiura, 1980; 1982a,b; Tohdoh and Sugiura, 1982; Zurawski et al., 1982). However, the genes presently mapped on the Nicotiunu ct genome can account for only about 20% of the Nicotiunu ct genome size of 160 kb (Shen et al., 1982). The function of the bulk of the ct genome is still unknown. One approach toward determining functions for regions of the ct genome requires identification of the location of the promoters. The ct genome contains sequences that show extensive similarity with promoter sequences identified in E. coli (Whitfield and Bottomley, 1983). Therefore, we initially undertook to screen the ct genome for promoters that function in E. coli. To accomplish this we used the promotercloning, gulK-expression plasmid pK0 1, construct-

24

ed by cloning into pBR322

galK gene of E. coli

the promoterless (McKenney

et al., 1981; Rosenberg

et al., 1983). In this vector exogenous promoters

can be readily positioned

the galK gene, activating et al.,

1981;

advantage cloned genome

its expression

Rosenberg

of this

restriction

fragments

which possess

and characterized to our knowledge,

et al.,

selection

prokaryotic

upstream

from

(McKenney

1983).

By taking

procedure,

we have

of the N. tabacum ct

promoter

This is,

the first report on the utilization promoters

contain

linear form of pK0 1. After trans-

of E. coli, 35 red colonies

formation

cloned promoters

to be stable after repeated galactose-Ap

were identified plating

AND METHODS

(a) Cloning

of chloroplast

DNA

by prokaryotes.

restriction

frag-

ments DNA was isolated from chloroplasts of N. tabacum var. MD609 (Rhodes and Kung, 1982), digested to completion with EcoRI, and ligated with the EcoRI-treated plasmid pKO1 (McKenney et al., 1981). The ligation mix (containing 2 pg/ml DNA) was used to transform E. coli N 100 (galK_ recA - ). The GalK’ transformants resistant to 30 pg/ml of Ap were selected on MacConkey-galactose-Ap agar. The red GalK’ colonies usually result from insertion of a promoter fragment into pKO1. (b) Southern hybridization ing

and nucleotide

sequenc-

Southern hybridization was performed as described by Zhu et al. (1982). Nucleotide sequencing was by the dideoxynucleotide method of Sanger et al. (1977), using M13mp8 and M13mp9 vectors (Bethesda Research Laboratories). Restriction fragments were sequenced in both directions.

RESULTS

(a) Cloning and selection

to

and found

on MacConkey-

plates.

(b) Identification

and mapping of promoter-active

fragments The pKO1

derivatives

purified

colonies fell into live size categories, insertion

of promoter-active

frag-

ments N. tabacum ctDNA fragments were “shot-gun” cloned into pK0 1. Approx. 40 fragments of ctDNA produced by EcoRI were randomly ligated to the

from the 35 red with the longest

larger than pK0 1 and the smallest less than

1 kb. In one case, two ctDNA MATERIALS

presumed

activity in E. coli,

several of these fragments.

of higher plant organelle

EcoRI-generated

fragments

were in-

serted (not shown). To determine whether all insertions were of ct origin, the inserts were excised with EcoRI and compared with EcoRI-digested ctDNA by agarose-gel electrophoresis. Each of the six cloned fragments was identical in size to one of the approx. 40 EcoRI fragments of ctDNA. The designation of each clone refers to the particular EcoRI fragment(s) of ctDNA present in the chimera. Hence, pKOl-4 contains a 4.5-kb fragment of ctDNA, pKOl-8 a 3-kb insert, pKOl-20, a 1.8-kb insert, pKOl-26, a 1.2-kb insert and pKOl-28-35 two inserts whose sum is 1.7 kb. 32P-labelled probes of EcoRI-generated promoteractive fragments were hybridized to BamHI-digested N. tabacum ctDNA. The locations of the promoteractive fragments were thus identified and mapped on the available BamHI restriction map of the ct genome (Fig. 1). Three promoter-active fragments were situated within the inverted repeat region, near the genes for rRNA. The other two were located in the large single-copy sequence. For example, the 4.5-kb and the 3.0-kb ctDNA inserts hybridized with BamHI fragments 5 and 13, respectively. These are adjacent fragments and are located in the inverted repeat regions (Fig. 1). They have been identified as EcoRI fragments 4 and 8, containing the genes for 23s and 16s RNAs (Sugiura and Kusada, 1979). The 1.2-kb insert in pK0 l-26 also hybridized with BamHI fragment 5. According to the physical map, the fragments in pKOl-4, pKOl-8 and pK0 l-26 covered almost the entire region for all four rRNA genes. The exact locations of these promoteractive fragments are illustrated in Fig. 2, which was constructed from the data of Takaiwa and Sugiura (1980; 1982a,b) and Tohdoh and Sugiura (1982). It is clear that pKOl-8, pKOl-4 and pKOl-26 contain

pKOl-28-35

Fig. 1. Location

of the promoter-active

identify the location pKOl-26

and pKOl-20

5 and 13. pKOl-28-35 adjacent

hybridized contains

on the ct genome.

that include

EcoRI fragments

of each promoter-active

the cistrons

to the respective

two EcoRI fragments,

The two regions

on the BarnHI

EcoRI fragment

BumHI fragments denoted

for the 4.5S, 5s. 23s and 16s ribosomal

the 16S, 23S, and 4.5s + 5S rRNA genes, respectively. The promoter-active fragment in pKOl-20 (1.8 kb) hybridized with the largest Bum HI fragment (21.5 kb), but its precise location within the larger fragment has not yet been determined. pKOl-28-35 (1.7-kb insert) contains two small ctDNA fragments that hybridized to two nonadjacent BumHI frag-

map of the N. tabacum ct genome.

map as determined

13.5 and 1. pKOl-4

and this plasmid

of the ct genome

restriction

on the BamHI

hybridized

hybridized

to two BamHI

by bars outside

by Southern

to the adjacent

fragments,

the figure represent

Hatched

hybridization. BumHI

areas

pKOl-8, fragments

28 and 20, which are not the inverted

repeat

regions

RNAs.

ments of the ct genome. Both EcoRI fragments appear to be essential for promoter activity, since cutting pKOl-28-35 with EcoRI followed by ligation and transformation generates red colonies that contain a plasmid indistinguishable from pK0 1-2835. Moreover, clones containing only fragment 28 or 35 remained galK- .

26

Ile

16s

Val

Ala

23s

*+-l

4.5s 5s

I

I

EcoRI

EcoRI

EcoRI

EcoRI

Hind III 4

EcoRI 1

lZWbp pKOl-26

1 TTGAGTTfCTCGACC -

TTGTCTATCGTTGGC -

TTGGGGCCTCACAAT

TTACGTCCGTACGTGCAC _

CTTTGACTTAGGAT

CTCTATGGTAGAAT

CACTAGCCAATATGCT

GCATCAGCGATATCGT

Fig. 2. Identification cloned restriction

of potential

fragment

The EcoRI fragment

promoter

corresponds

in pKlO-8

includes

of the tRNAVa’ gene exists a putative

sequences

to a previously

mapped

the gene encoding

prokaryotic-like

within the tRNAAla gene. In the EcoRI fragment

and manipulation

in pKOl-8,

region of the ct genome

pKOl-4

spanning

tRNAVa’ and a major portion

promoter.

and a major portion ofthe 23s rRNA gene. A putative

(c) Characterization

in the N. tabacum ctDNA

The EcoRI fragment

prokaryotic-like

in pKOl-26,

promoter

two potential

of promoter-

active fragment E-26

The 1200-bp EcoRI fragment E-26, present in pKOl-26, contains a single Hind111 site, which separates E-26 into two smaller segments of 496 bp and 704 bp (Fig. 3A). To determine the location of the promoter(s) in E-26, pKOl-26 was digested with Hind111 and religated. This deletion removes the 704-bp HindIII-EcoRI region of E-26 plus a 290-bp EcoRI-Hind111 fragment of pKO1 which is located upstream from the gulK gene (Fig. 3B). Transformation of E. coli with this deleted plasmid generated guZK + colonies. These results indicate the presence of at least one promoter in the 496-bp EcoRI Hind111 region of E-26. The 994-bp Hind111 fragment deleted from pK0 l26 was ligated to HindIII-digested pK0 1. This generated pKOl-26-S (Fig. 3C), in which the 704-bp ctDNA is in the same orientation as in pKOl-26. Since pKOl-26-S generated gaZK’ colonies, a promoter must also exist in the 704-bp HindIII-EcoRI region of E-26. To determine whether the 704-bp HindIII-EcoRI region of E-26 would promote galK expression in the reverse orientation, the 704-bp fragment was eluted from an agarose gel and ligated to pKO1 previously cut with EcoRI and Hind111 (Fig. 3D). The ligation mix was used to transform E. coli to ApR, and in all the transformants examined guZK remained unex-

16s rRNA.

spans the tRNA”’

can be identified

prokaryotic-like

(heavy lines). Each

coding for tRNA and rRNA.

of the gene encoding

in pKOl-4

sequence

and pKOl-26

introns

promoter

in the sequence sequences

Upstream

and tRNAA’” genes of the intron

can be identified.

pressed. Analysis of plasmids from three transformants demonstrated that each contained a 704-bp fragment that could be released by cleavage with EcoRI and HindIII. These data suggest that the 704-bp fragment contains a promoter that is active in one orientation only. (d) Sequence analysis ments

of the promoter-active

frag-

As can be seen from Fig. 2, the 3054-bp EcoRI fragment inserted in pKOl-8 contains the genes of tRNA”“’ and 88.9% of the gene for 16s rRNA (Tohdoh and Sugiura, 1982). There is a single EcoRI site in the 16s rRNA gene which cleaves the 1542-bp gene into 1372-bp and 170-bp segments (Tohdoh and Sugiura, 1982). Similarly, the 4450-bp EcoRI fragment inserted in pKOl-4 contains the genes for tRNAne, tRNAAla and 78.5% of the 23s rRNA (Takaiwa and Sugiura, 1982a). The 23s rRNA gene also contains a single EcoRI site, and therefore a 604-bp segment of this gene is missing from the ctDNA in pKOl-4. It should be noted that both the tRNA”” and tRNA*‘” genes contain a large intron of 710 and 750 bp, respectively (Takaiwa and Sugiura, 1982a). Since the tRNA”“l, tRNA”“, tRNA*‘“, and 16S, 23s and 4.5s rRNA genes are transcribed as a large precursor (Hartley and Head, 1979), there is a putative promoter region upstream of the tRNA”“l in pKOl-8. We presume this pro-

21

E-26

A.

Hind III

EcoRI

EcoRI I

496

704 Hind III

6.

C.

gal K E+H i

D.

gal K Fig. 3. Subcloning

of the promoter-active

site, which cleaves it into two unequal plasmid

pKOl-26

contained

was generated.

the 496-bp subfragment

994-bp Hind111 fragment 704-bp ct fragment between

EcoRI fragment size subfragments

(C) Digestion

of pKOl-26

of E-26 in a derivative

(704 bp + 290 bp) from pKOl-26

relative togulK

was accomplished

E-26 from pKOl-26.

with Hind111 and religation

generated

contains

into pKO1

generated

by digesting pKOl-26-S pKOl-26-S-R.

pKOl-26-S.

a recombinant (D) Reversal

with Hind111 andEcoR pKOl-26-S-R,

a single Hind111

into pKO1 the recombinant 4.1-kb plasmid

of pKO1 deleted for the 290 bp between EcoRI and HindIII.

the EcoRI and Hind111 sites of pKO1. This generated

in E. coli.

(A) The 1.2-kb E-26 fragment

of 496 and 704 bp. (B) When E-26 was inserted

Insertion

of the orientation

and inserting

unlike pKOl-26-S,

which of the of the

the 704-bp fragment did not express gulK

28

moter provides the promoter activity of this EcoRI fragment when it is inserted in pKO1. Likewise, the promoter region upstream of this 5s rRNA is presumed to be responsible for the promoter activity exhibited in E. coZiby fragment E-26 in pKOl-26. Nucleotide sequence analysis of the first 120 bp of the 496-bp EcoRI-Hind111 fragment of E-26 confirmed the previously published sequence data for this region, and demonstrated the occurrence of a potential -35 sequence, TTGGGG, followed by 19 bp downstream by a potential -10 sequence (Pribnow, 1975) TATGCT (see Fig. 2). However, no promoter region in the 4450-bp EcoRI fragment of pKO14 was reported. The observed promoter activity in this fragment must be conferred by a structure closely resembling the promoter sequence that can be recognized by E. coli. After a careful examination, the following sequence was identified in the intron of tRNAA’“: ---~GT~TATCG~GGC~T~TATGGTAGAAT--- (Takaiwa and Sugiura, 1982a). This sequence approximates the E. coli consensus promoter at both the -35 and -10 recognition sites (underlined), TTGACA and TATAAT, respectively (Rosenberg and Court, 1979; Hawley and McClure, 1983). Thus, in each of three EcoRI fragments inserted into pKO1 a putative promoter region can be recognized. Fragment E-26 from pKOl-26 was shown to contain at least two promoters, one in the 496-bp ~~~dIII-~c~RI segment and the other in the 704-bp HindIII-EcoRI segment. The sequence analysis of the 704-bp segment showed that at approx. 180 bp upstream from the EcoRI site there is a promoterlike sequence ---TTGCTGGTGGCTAACGTATACCCCTGTAGCGT. This sequence also resembles -I__ the consensus sequence of prokaryotic promoters and, therefore, presumably can be recognized in E. coli. No promoter sequence was identified in the reverse orientation of this segment, and the 704-bp fragment has been shown to exert promoter activity in only one orientation.

DISCUSSION

Our results support the notion that promoters which function in prokaryotes are present within the Nicotiana ct genome (Whitfeld and Bottomley, 1983).

This concept was previously advanced, primarily based on the observation that conserved sequences upstream of tr~s~~ption start sites of ct genes resemble prokaryotic promoters (Rosenberg and Court, 1979; Hawley and McClure, 1983). However, whether such promoter sites actually function in the chloroplast is not known. The results presented here provide the first experimental evidence that the putative ct promoters identified by nucleotide sequence analysis can function in controlling the expression of genes in E. coli. Our sequence analysis confirms the previously reported putative promoter sites, as well as identifies new consensus sequences in the promoter-active fragments. Indeed, reversal of the orientation of one conserved promoter sequence abolished the promoter activity, and upon examination of the DNA sequence data no prokaryotic promoter-like sequence could be identified in the reverse o~entation. Thus, a close st~~ture-function relationship of those conserved sequences is indicated. Table I lists the conserved sequences around the putative promoter regions of a number of Nicotiana ct genes (Shinozaki and Sugiura, 1982; Takaiwa and Sugiura, 1980; 1982a,b; Tohdoh and Sugiura, 1982; Zurawski et al., 1982). It is interesting to note the remarkable similarities between the promoter regions of E. coli and Nicotiana ct rRNA genes. Within the -35 region, the highly conserved sequence of the trimer -TTG- in -TTGACA- is found in all Nicotiana ct promoter-active fragments. The -ACAsequence is somewhat less conserved, as is the case with E. coli. On the other hand, three of the six base pairs in the - 10 region hexamer are highly conserved. The so-called “invariant 2”’ of the -TATAAT Pribnow (1975) box is present in all cases. In addition, the lirst two bases, TA, of the Pribnow box are also highly conserved. It should be noted that in all cases found in the Nicotiana et genome, the spacing between these two hexamers is nearly 17 bp. Whether the degree of variation of those consensus structures is reflected by the degree of effectiveness as promoters has not been investigated. There seems little doubt that the prokaryotic promoter-like sequences in ctDNA function as promoters in E. coli. What is most interesting is our detection of a sequence in the intron of tRNAAla which appears to be recognized and utilized by E. coli as a promoter (Table I). Although the occur-

29

TABLE

I

Nucleotide Source

sequences

of putative

prokaryotic-like

of promoter

E. cd consensus

promoter

Promoter

regions

in Nicotiuna ctDNA

region

References

-35

Spacer

-10

TTGACA

J7( * 2)

TATAAT

sequence

Hawley

and McClure

Rosenberg Nicotiana tRNAVa’ - 16s RNA

TTGAGT

17

TAGGAT

Tohdoh

et al. (1981)

Nicotiana 5S rRNA

T TGGGG

19

TATGCT

Takaiwa

and Sugiura

Nicotiana tRNAAS”

TTGGGA

11

TATAAT

Kato et al. (1981)

Nicotiana tRNA”“’

TAGATT

19

TATGAT

Deno et al. (1982)

TTGCTT

18

TATAAT

TTGATG

17

TATCTT

TTGACA

17

TAAAAT

Nicotiana tRNAVa’

(1983)

and Court (1979) (1980)

Deno et al. (1982)

Nicotiana rbcL”

TTGCGC

18

TACAAT

Shinozaki

and Sugiura

Nicotiana psbAb

TTGACA

17

TATACT

Zurawski

et al. (1982)

TTGTCT

17

TAGAAT

Takaiwa

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