JOURNAL OF BACTERIOLOGY, Apr. 1990, p. 2178-2180 0021-9193/90/042178-03$02.00/0 Copyright © 1990, American Society for Microbiology

Vol. 172, No. 4

Regulatory Elements of the Raffinose Operon: Nucleotide Sequences of Operator and Repressor Genes CHARALAMPOS ASLANIDISt AND RUDIGER SCHMITT*

Lehrstuhlffur

Genetik, Universitat Regensburg, D-8400 Regensburg, Federal Republic of Germany Received 10 July 1989/Accepted 21 December 1989

The raffinose (raf) operon is negatively controlled by the specific binding of raf repressor (raiR gene) to raf operator (rafO) DNA. Both ra.R and rafO have been sequenced. The 1,011-base-pair raJR gene encodes a 336-amino-acid polypeptide containing an N-terminal helix-turn-helix motif. rafO, as defined by in vivo titration of raf repressor, consists of two nearly identical 18-base-pair palindromes that flank the -35 box of the raf promoter.

The genetic organization of the plasmid-borne raffinose

(rap operon with three recently sequenced (2) genes encod-

ing a-galactosidase (rafA), Raf permease (raJB), and sucrose hydrolase (rafD) and their negative control by a repressor (the raJR gene) bear close resemblance to the organization and control of the Escherichia coli lactose (lac) operon (8). However, the specificities of lac and raf gene controls are quite distinct; the latter has been conserved in 25 raffinose plasmids from different sources (15). To elucidate the molecular basis of this regulatory specificity, we have sequenced rafR and the adjacent raf regulatory region of plasmid pRSD2 (2, 4) and have identified two palindromic operator sequences by in vivo titration experiments. A 1,289-base-pair HindIII-PstI fragment containing rafR and rafO from pRSD2 was subcloned into vector pUC8 (19) and sequenced by the chain termination method (12) as described before (2, 7). The nucleotide sequence presented in Fig. 1 contains one large 1,011-base-pair open reading frame with a derived 336-amino-acid sequence (calculated molecular weight, 36,700) shown below the DNA sequence. Its identity with the repressor gene, raf?, is suggested by the following observations. (i) Transformation of the raf-constitutive E. coli DS338-2 (containing a chromosomal insertion of Arafl rafO+A+ [17]) with the cloned HindIII-ScaI fragment containing the open reading frame (Fig. 1) led to inducible synthesis of a-galactosidase. (ii) In accord with the derived primary structure (Fig. 1), the N-terminal sequence of purified raf repressor reads Ser-Leu-Lys-Ala-Ile (C. Aslanidis, I. Muiznieks, and R. Schmitt, submitted for publication). The absence of an amino-terminal methionine suggested posttranslational processing (18). (iii) By primary structure comparisons with other DNA-binding proteins (10, 13, 14), a helix-turn-helix motif (Fig. 1, ao and a2 helices underlined) has been identified in the amino-terminal portion of the derived peptide sequence. The recognition helix a2 iS thought to specifically interact with palindromic operator sequences, as has been shown in other repressor-operator systems (1, 9, 11, 13). (iv) A spontaneous mutation that led to constitutive expression of the raf operon (16) was located within the open reading frame by sequencing. This C-to-A transition at position 314, which resulted in a nonconservative exchange of Ala-50 to Glu-50 (Fig. 1), rendered the raf repressor inactive. * Corresponding author. t Present address: Lawrence Livermore National Laboratory, Livermore, CA 94550.

2178

By correlation with consensus sequences and their spacing (6), we have assigned presumptive promoter signals and a ribosome-binding site upstream of the rafR translation start (Fig. 1). However, since Si mapping did not yield detectable bands (because of low representation of raft messenger), definite assignment of the promoter must await deletion mapping and mutant analysis similar to an approach chosen to define the weak lacI gene promoter (5). The nucleotide sequence downstream of rafR contains the previously identified promoter and ribosome-binding site of the raf structural genes (2) and two nearly identical 18base-pair palindromic sequences flanking the -35 promoter box (Fig. 1). These symmetrical sequence elements were

identified as the raf operator (rafO) by a set of in vivo repressor titration experiments. Cells of E. coli C600 (3) that contained the entire raf operon on the single-copy prophage

PlKmRaf (2) were transformed with high-copy pUC8 (19) recombinants bearing four different fragments from the rafR-rafA intercistronic region, as shown in Fig. 2. Whenever the recombinant plasmid contained a functional operator sequence, raf repressor molecules were scavenged, leaving the resident operator unoccupied. The resulting expression of the prophage-borne raf operon was monitored

by measuring ox-galactosidase activity (16). The ability or inability (+ or -) of each fragment to bind raf repressor is diagrammed in Fig. 2. A 169-base-pair PstI fragment (clone pRU645), spanning the entire rafR-rafA intercistronic region (Fig. 1), and three subclones, pRU950, pRU951, and pRU952, were probed. Repressor binding was observed with pRU645 and with pRU952, both of which contain the central 44-base-pair Sau3A fragment, but not with the two flanking sequences borne by pRU950 and pRU951 (Fig. 2). These results confined the raf operator to two 18-base-pair palindromic sequence elements, termed 01

and 02, that occupy most of the Sau3A fragment. Distinctive interactions between operator half-sites with the consensus AXCCGAAAC (Fig. 2) and the a2 recognition helix of the repressor (Fig. 1) are thought to be responsible for the specificity of raf regulation analogous to related control systems (9, 13). The conspicuous location of the -35 raf promoter box between the two repressor-binding sites may be essential for the efficient control of transcription initiation. The genetic organization of the 6.1-kilobase-pair raf operon and the gene-function relationships of one regulatory and three structural genes, as elucidated in this and a previous study (2), are summarized in Fig. 3. This includes

1 101

1 201 13

AAGCTTATTATGCTTCCATCGGAAACAATGATCTGGCAGCCTGAGTTCACAGATAAAATATCTCTCCAGGAAACCCGGGGCTGTTCATCATGCAAGTCTG

* . (SD) (-10) (-35) 3r a fERK TCGATTACTGGCTGGTGACGGAATTTTCTGGATTTCCGGCTTAGAACCACAGCAGGAGATAATATGTCACTTAAAGCGATTGCCACGACACTCGGTAT M S L K A I A T T L G I

301 46

TTCTGTCACCACTGTCAGTCGGGCTCTTGGAGGCTTTTCAGATGTGGCTGCTTCTACCCGTGAGCGCGTGGAAGCGGAAGCACGTCGACGAGGTTACCGC S V T T V S R A L G G F S D V A A S T R E R V E A E A R R R G Y R A OZ A CCTAATACACAGGCAAGAAGACTCAAAACCGGTAAAACCGATGCTATCGGTCTGGTTTATCCTGAAAATGATGTGCCGTTTAACAGCGGTGTTTTTATGG P N T Q A R R L K T G K T D A I G L V Y P E N D V P F N S G V F M

401 79

ATATGGTCAGTTGCATCAGCAGGGAACTTGCTTATCATGATATTGACTTACTGCTGATCGCTGATGATGAGCATGCAGACTGCCACAGCTATATGCGGCT D M V S C I S R E L A Y H D I D L L L I A D D E H A D C H S Y M R L

501 113

TGTTGAAAGTCGCAGAATTGATGCTCTTATCATTGCACATACTCTGGATGACGATCCCCGTATCACACATCTTCATAAAGCAGGTATTCCGTTTCTGGCT V E S R R I D A L I I A H T L D D D P R I T H L H K A G I P F L A

601 146

CTTGGACGGGTACCGCAGGGCTTGCCCTGTGCGTGGTTTGACTTTGATAATCATGCCGGAACCTGGCAGGCAACCCAGAAGCTGATTGCTTTGGGACATA

701 179

AGAGTATTGCGCTGTTGAGCGAGAACACTTCACATTCTTATGTTATTGCAAGACGTCAGGGATGGCTTGATGCACTGCATGAGCATGGACTGAAAGATCC K S I A L L S E N T S H S Y V I A R R Q G W L D A L H E H G L K D P

801 213

ATTGTTGCGGCTGGTTTCTCCCACGCGACGAGCGGGCTATCTGGCTGTGATGGAGTTAATGTCATTACCGGCGCCACCAACAGCTATTATTACTGACAAT L L R L V S P T R R A G Y L A V M E L M S L P A P P T A I I T D N

901

246

GACCTGAGTGGAGATGGTGCGGCTATGGCGCTGCAGTTGAGAGGGCGTCTTTCAGGGAAAGAAGCTGTATCTCTGGTTGTATATGATGGTTTGCCTCAGG D L S G D G A A M A L Q L R G R L S G K E A V S L V V Y D G L P Q

1001

ACAGCATTATTGAGCTGGATGTGGCTGCTGTTATTCAGTCAACACGAAGTCTCGTTGGTCGTCAGATTTCTGACATGGTGTATCAGATAATCAATGGTGC D S I I E L D V A A V I Q S T R S L V G R Q I S D M V Y Q I I N G A

279

L

G

R

V

P

Q

G

L

P

C

A

W

F

D

F

D

N

H

A

G

T

W

Q

A

T

Q

K

L

I

A

L

G

H

1101 313

ATCACCAGAATCACTGCAGATAACCTGGACACCGATATTTTACCCTGGTAGCACGGTTCATTCTCCTTCCTTCTGATTTTTTATCCAGATCACACAACCG S P E S L Q I T W T P I F Y P G S T V H S P S F * x,e:E Ca OLE

1201

AAACGTTTTGGTTGATG-TT-C-G-AAA-C-G m-C-GG-A-TCAACAGTAAGACATACCTGAAAGCGGAGATGTCTTATGATTTCAAAGTACTgcag -

w.

35

-1 0

N

SD

I

S

K

Y

C

FIG. 1. Sequence of a 1,289-nucleotide HindIII-PstI fragment (with complete terminal recognition sequences) from pRSD2 (4) containing the rafR gene and flanking regions. The deduced amino acid sequences of the repressor protein and the presumptive operator-binding helices, a, and a2 (underlined), are shown below the nucleotide sequence. Numbers on the margin run sequentially for nucleotides (upper line) and amino acid residues (lower line). The translational start of rafR, presumptive promoter boxes (-35, -10), and a ribosome-binding site (SD) are indicated. Also shown are the raf operator, rafO, characterized by palindromic sequences (dashed lines), and the translation start of rafA together with previously assigned transcription and translation signals (2). A spontaneous raf-constitutive mutant (16), identified as a C-to-A transversion at position 314 in rafR that leads to an Ala-50-to-Glu-50 exchange, is shown. The sequence has been submitted to the GenBank data base; its accession number is M29849.

01

JGATCACAC

IAACCGAAACGTTTT

pRU950

02

-35

GGTAGC

GAT

^. pRU952

Z.-

pRU951

pRU645 ® FIG. 2. Identification of raf operator by in vivo repressor titration. E. coli C600 (3) containing the wild-type raf operon on a single-copy plasmid (PlKmRaf [2]) was transformed with pUC8-derived (19) high-copy recombinant plasmids bearing the presumptive raf operator (rafO) sequence. A 169-base-pair PstI fragment of pRSD2 (2, 4), which contains the entire rafR-rafA intercistronic region, and three subfragments (nucleotides numbered as in Fig. 1) were ligated into pUC8 and used for repressor titration. As presented here, the right-hand Sau3A cloning site of pRU952 was preserved by ligation into the BamHI site of pUC8 (19). The Sau3A cleavage sites are marked (A). The presence (+) or absence (-) of a-galactosidase synthesis (16) is indicated for each fragment. Accordingly, the two nearly identical 18-base-pair palindromes (boxed) contained on the central 44-base-pair Sau3A fragment are the presumptive operator sequences, termed 01 and 02. Note the conspicuous arrangement of the operator sequences and the -35 box. 2179

2180

J. BACTERIOL.

NOTES t

5t rofR


a-GaaLctosiduse 4x81

r~~~~~

Permease 1x47

-

4

,1kbp

~~ofB 8 rof D

Scr

Hydrolose 2x54

00 ~~~~~~~~~~~~0

{

FIG. 3. Overview of gene-function relationships in the 6.1-kilobase-pair (kbp) raf operon. The arrangement of regulatory (raf?) and structural genes (rafA, rafB, and raJD), their transcripts, transcription regulation via induction (+I and -I), and the configurations of functional gene products with their subunit molecular masses (in kilodaltons) are shown. 0, Operator; P, promoter; Scr, sucrose.

the negative mode of transcription control by the specific binding of tetrameric rafrepressor molecules (R. Jaenicke, I. Muiznieks, C. Aslanidis, and R. Schmitt, FEBS Lett., in press) to raf operator DNA and the dissociation of this complex by the addition of inducer. In vitro binding and footprinting experiments with purified repressor that confirm this mode of control (Fig. 3) will be presented in a subsequent report (Aslanidis et al., in preparation). We thank Peter Heinrich for technical advice, Anneliese Mitterer for artwork, and Edwin Pleier for help in preparing the manuscript. This investigation was supported by the Deutsche Forschungsgemeinschaft. LITERATURE CITED 1. Anderson, J. E., M. Ptashne, and S. C. Harrison. 1987. Structure of the repressor-operator complex of bacteriophage 434. Nature (London) 326:846-852. 2. Aslanidis, C., K. Schmid, and R. Schmitt. 1989. Nucleotide sequence and operon structure of plasmid-borne genes mediating uptake and utilization of raffinose in Escherichia coli. J. Bacteriol. 171:6753-6763. 3. Bachmann, B. J. 1983. Linkage map of Escherichia coli K-12, edition 7. Microbiol. Rev. 47:180-230. 4. Burkhardt, H. J., R. Mattes, K. Schmid, and R. Schmitt. 1978. Properties of two conjugative plasmids mediating tetracycline resistance, raffinose catabolism and hydrogen sulfide production in Escherichia coli. Mol. Gen. Genet. 166:75-84. 5. Calos, M. P. 1978. DNA sequence for a low-level promoter of the lac repressor gene and an 'up' promoter mutation. Nature (London) 274:762-766. 6. Harley, C. B., and R. P. Reynolds. 1987. Analysis of E. coli promoter sequences. Nucleic Acids Res. 15:2343-2361. 7. Heinrich, P. 1986. Guidelines for quick and simple plasmid sequencing. Boehringer GmbH, Mannheim, Federal Republic of

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Germany.

8. Jacob, F., and J. Monod. 1961. Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3:318-356. 9. Lehming, N., J. Sartorius, M. Niemoller, G. Genenger, B. v.

19.

Wileken-Bergmann, and B. M{iler-Hill. 1987. The interaction of the recognition helix of lac repressor with lac operator. EMBO J. 6:3145-3153. Matthews, B. W., D. H. Ohlendorf, W. F. Anderson, and Y. Takeda. 1982. Structure of the DNA-binding region of lac repressor inferred from its homology with cro repressor. Proc. Natl. Acad. Sci. USA 79:1428-1432. Ohlendorf, D. H., W. F. Anderson, R. G. Fischer, Y. Takeda, and B. W. Matthews. 1982. The molecular basis of DNA-protein recognition inferred from the structure of cro repressor. Nature (London) 298:718-723. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. Sauer, R. T., and C. 0. Pabo. 1984. Protein-DNA recognition. Annu. Rev. Biochem. 53:293-321. Sauer, R. T., R. R. Yocum, R. F. Doolittle, M. Lewis, and C. 0. Pabo. 1982. Homology among DNA-binding proteins suggests use of a conserved super secondary structure. Nature (London) 298:447-451. Schmid, K., S. Ritschewald, and R. Schmitt. 1979. Relationships among raffinose plasmids determined by the immunochemical cross-reaction of their a-galactosidases. J. Gen. Microbiol. 114:477-481. Schmid, K., and R. Schmitt. 1976. Raffinose metabolism in Escherichia coli K12. Purification and properties of a new oa-galactosidase specified by a transmissible plasmid. Eur. J. Biochem. 67:95-104. Schmitt, R., R. Mattes, K. Schmid, and J. Altenbuchner. 1979. Raf plasmids in strains of E. coli and their possible role in enteropathogeny, p. 199-210. In K. N. Timmis and A. Puhler (ed.), Plasmids of medical, environmental and commercial importance. Elsevier/North Holland Publishing Co., Amsterdam. Sherman, F., J. W. Stewart, and S. Tsunasawa. 1984. Methionine or not methionine at the beginning of a protein. Bioessays 3:27-31. Vieira, J., and J. Messing. 1982. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 33:259-268.