Feb 24, 1983 - hibition of septum formation (Kirby et al., 1967), induction of prophage ... genes (Rosenberg and Court, 1979). Only one possible Prib-. HincIl.
The EMBO Joumal Vol.2 No.5 pp.787-789, 1983
Nucleotide sequence of promoter, operator and amino-terminal region of caa, the structural gene of colicin A Juliette Morlon, Roland Lioubes, Martine Chartier, J.Bonicel and Claude Lazdunski* Centre de Biochimie et de Biologie Mol&ulaire du C.N.R.S., 31 Chemin Joseph Aiguier, B.P. 71, 13402 Marseille Cedex 9, France Communicated by B.A.D.Stocker Received on 24 February 1983
The nucleotide sequence of 378 bp covering the promoteroperator regions and the region coding for the N-terminal portion of the colicin A gene was determined. These assignments were made possible by the determination of the N-terminal 12 amino acids of the colicin A protein. DNA sequence homologies between operator regions of recA, lexA, uvrA, uvrB, cea and caa genes are discussed. Key words: colicin A/SOS functions/nucleotide sequence Introduction The synthesis of colicin A, an antibacterial protein, is repressed in Citrobacterfreundii or Escherichia coli cells harboring the plasmid ColA (pColA) under ordinary conditions. Upon treatment with u.v. light or chemicals such as mitomycin C, the synthesis of colicin is induced. This induction is considered as one of the 'SOS responses' (Little and Mount, 1982) occurring after DNA damage or inhibition of DNA replication in E. coli. These responses include enhancement of repair capacity (Clark and Margulies, 1965; HowardFlanders and Boyce, 1966; Tomizawa and Ogawa, 1968), inhibition of septum formation (Kirby et al., 1967), induction of prophage replication (Brooks and Clark, 1967; Herman and Luria, 1967; Devoret, 1981), RecA protein synthesis (Gudas and Pardee, 1976) and mutagenesis (Miura and Tomizawa, 1968). Colicin induction also appears to be one of the 'SOS responses'. However, only the case of colicin El has been documented so far (Ebina et al., 1982). It would, therefore, be useful to provide more information on this system. In previous work, we have made a restriction map of the plasmid ColA (Morlon et al., 1982a) and determined the localization of the structural gene for colicin A activity (caa) in this map (Morlon et al., 1982a). Here, we present the DNA sequence involved in the regulation of caa gene expression. The assignments for promoter-operator regions and coding sequences were made possible by the determination of the sequence of the first 12 amino acid residues of the aminoterminal part of colicin A protein. Results and Discussion We had previously shown that the gene caa is entirely included in the 2.17-kb HincII fragment of plasmid ColA (Morlon et al., 1982b). Two PstI sites are located in this fragment (Morlon et al., 1982a). Assuming that the direction of caa transcription was from the 3.68-kb HincII site to the 3.27-kb PstI site (Morlon et al., 1982a), we sequenced this HincII-PstI fragment. Thus, a physical map of this fragment *To whom reprint requests should be sent.
© IRL Press Limited, Oxford,
England.
was constructed using the restriction endonucleases HpaII, DdeI, AvaII, Sau3A (Figure 1) and its nucleotide sequence was determined by the chemical modification method of Maxam and Gilbert (1980). Figure 1 shows the strategy for sequencing this fragment. The established nucleotide sequence is presented in Figure 2. In the 378 bp upstream from the PstI site there is only one translation initiation codon ATG followed by a nucleotide sequence fitting the N-terminal amino acid sequence of colicin A that we found to be: Pro-Gly-Phe-Asn-Tyr-Gly-Gly-LysGly-Asp-Gly, after determination by automatic Edman degradation of this protein. The N-terminal methionine was not found in the purified colicin A, a case often encountered in E. coli (Waller, 1963). The above finding confirms our previous suggestion that colicin A has no cleavable signal sequence in the N-terminal portion (Varenne et al., 1981). Our previous observation that, in contrast to exported proteins of E. coli, colicin A is produced in free polysomes and not in membrane-bound ones is also consistent with this result (Varenne et at., 1981). About 6 bp upstream from the translation initiation codon, there is the sequence AGGA, complementary to the 3' end of 16S RNA that serves as a ribosome-binding site (Shine and Dalgamo, 1974). This so-called 'Shine-Dalgarno' sequence has been found in all analyzed prokaryotic sequences at a distance of 4-9 nucleotides preceding the ATG codon (Rosenberg and Court, 1979). Regulatory sequence of caa The nucleotide sequence upstream from the translation start point was analyzed for sequences which could be involved in the interaction with RNA polymerase and which are known to be rather well conserved in promoters of E. coli genes (Rosenberg and Court, 1979). Only one possible PribU -~~~
0
L
~~~
-
uiWZO
x x
,
100
-
0
fl4U
~~I,11 I 200
U
300
HincIl Avail
. 4 b
Sau 3A
Hpall Dde
Pst I
Fig. 1. Restriction map and sequencing strategy of the 378-bp HincII-PstI fragment of ColA DNA. Distances are given in kbp from the HincIl site. Horizontal arrows below the map indicate the position, direction and length of the sequence determined for each fragment.
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J.Morlon et al.
OpTA
TCA Tr4r TAC TGT ATA TAA ACA CAT GTG AAT ATA TAC AGT TTT TGG TGT GGC AGA GCA CTT GAC AGC ATG GAA CTG CCG GGC GTA CTG TCG TAC CTT GAC GGC CCG CAT CAT ACT AAMA ATG ACA TAT ATT TGOT GOA CAC TTA TAT ATG TCA AAA ACC ACA CCG TCT CGT GAA I 60 I 30 ! 0 41e1t Pro ATA ACA GCG TGG CGG CAG GTA GCC GCN CAG CAA TAA ACC GCA CAC CAG ATT TCT CAT AAA ACA AAA ATA AAA Ar. G7A AAG ATT TAT TGT CGC ACC GCC GTC CAT CGG CGN GTC GTT ATT TGG CGT GTG GTC TAA AGA GTA TTT TGT TTT TAT TTT TCf CCtr TTC TAA ! 150 ! 120 ! 90
[AYqGl TAd
CCT GGA
! 23 ! 13 Phe Asn Tyr Gly Gly Lys Gly Asp Gly Thr Gly Trp Ser Ser Glu Arg Gly Ser Gly Pro Glu Pro Gly Gly Gly Ser His Gly Asn GGA TTT AAT TAT GGT GGA AAA GGT GAT GGA ACC GGC TGG AGC TCA GAA CGT GGG AGT GGT CCA GAG CCG GGT GGT GGT AGC CAT GGA AAT CCT AAA TTA ATA CCA CCT TTT CCA CTA CCT TGG CCG ACC TCG AGT CTT GCA CCC TCA CCA GGT CTC GGC CCA CCA CCA TCG GTA CCT TTA I 180 ! 240 ! 210 ! 3
Gly
! 53 ! 43 ! 33 Ser Gly Gly His Asp Arg Gly Asp Ser Ser Asn Val Gly Asn Glu Ser Val Thr Val Met Lys ??? Gly Asp Ser Tyr Asn Thr Pro Trp AGT GGT GGG CAC GAT CGT GGA GAT TCT TCC AAC GTA GGT AAT GAG TCT GTG ACG GTA ATG AAA CNA GGG GAT TCG TAT AAC ACC CCG TGG TCA CCA CCC GTG CTA GCA CCT CTA AGA AGG TTG CAT CCA TTA CTC AGA CAC TGC CAT TAC TTT GNT CCC CTA AGC ATA TTG TGG GGC ACC ! 330 ! 300 ! 270 ! 63 Gly Lys Val Ile Ile Asn Ala Ala GGA AAA GTC ATC ATC AAT GCT GCA CCT TTT CAG TAG TAG TTA CGA CGT ! 360 Fig. 2. Nucleotide sequence of the promoter-operator regions and the region coding for the amino terminus. Numbering of the nucleotides starts at 0, which corresponds to the HincII cleavage site. The amino acids deduced from the nucleotide sequence are placed above the nonsense strand. The heptanucleotide of the Pribnow box is framed in dashed lines, the inverted repeat sequence of the putative operator is underlined by arrows, the ribosome binding site is framed and the translation initiation codon is boxed twice. sequence was found, viz., TATCATT, forming nucleotides 23-29 (Figure 2); this sequence closely resembles that of the prototype promoter proposed (Rosenberg and Court, 1979). Since RNA polymerase is expected to recognize the 35-bp upstream locus and to bind to the Pribnow box locus 10-bp upstream from the start point of transcription, the latter should occur around nucleotide 39 (Figure 2). We will try to confirm this by in vitro transcription and sequencing of RNA transcripts. The LexA protein acts as a repressor for colicin El synthesis (Ebina et al., 1982). A similar repression could be expected to occur for caa expression. Just after the Pribnow box, an inverted repeat sequence (34-66) was found which closely resembles that of the putative operators of recA (Horii et al., 1980), lexA (Miki et al., 1981), uvrA (Kenyon and Walter, 1981), uvrB (Sancar et al., 1982) and cea genes (Ebina et al., 1981) that have LexA protein as repressor (Figure 3). In all these operators, one can observe one A-T pair framed by two G-C pairs (Figure 3 hatched area) which appears to be the minimal consensus sequence in agreement with that previously proposed. The TAT sequence is also highly conserved since it was found in all but one case, uvrB where TTT was found. Like most repressors, LexA protein should bind to operators have dyad symmetries shown in Figure 3, thereby excluding RNA polymerase from the promoter. However, the lexA repressor binds to different operators with different affinities (Little and Mount, 1982). It is striking to observe that a close similarity exists between operators of related genes like cea and caa or uvrA and uvrB; recA and le,xA promoters being significantly different from others. Out of 34 nucleotides in operators from cea and caa, there are only eight differences which means that -760o of the nucleotides were conserved. This conservation is even higher because, in the operator of caa, the AC sequence has been replaced by CA but equivalent changes in the opposite limb now
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Fig. 3. Comparison of possible structures of operators of recA (Horii et al., 1980), lexA (Miki et al., 1981), uvrA (Kenyon and Walter, 1981), uvrB (Sancar et al., 1982), cea (Ebina et al., 1982) with that of caa. The hatched circles presents the minimal consensus sequence. Homologous nucleotides present in all operators are shown in dotted circles. Homologous nucleotides found in different, but not all, operators are shown in white circles. Bars indicate positions of perfect inverted repeats.
(TG- GT) occurred. The lexA operator could be formed of a double inverted repeat. The second possible inverted repeat very closely resembles the operator of uvrA. The operator of lexA seems to have appeared as a duplication of other operators (those from recA, uvrA or uvrB). It will be of interest to further study the interaction of all these operators with LexA protein and to test the structures proposed which remain hypothetical. The rest of the nucleotide sequence of caa is presently being determined. Its comparison with the recently reported nucleo-
Nudeotide sequence of the regulating region of the caa gene
tide sequence of cea (Yamada et al., 1982) will be of interest. Materials and methods Chemicals The commercial source of restriction endonucleases and T4 polynucleotide kinase was Bethesda Research Laboratories. Lambda phage DNA, used as a calibration mixture in agarose gel electrophoresis, was purchased from Boehringer. [-y-32P]ATP (7000 Ci/mmol) was obtained from Amersham. Preparation of plasmid DNA and DNA sequence analysis The ColA plasmid originating from C. freundii CA31 was purified from strain E. coli W3 110 after amplification in the presence of chloramphenicol as previously described (Morlon et al., 1982a). Nucleotide sequence analysis of DNA was performed as described by Maxam and Gilbert (1980). Restriction fragments were separated in 4%7o polyacrylamide gel electrophoresis, treated with bacterial alkaline phosphatase, phosphorylated with [y-32P]ATP using T4 polynucleotide kinase and subjected to strand separation or secondary restriction cleavage. Determination of N-terminal amino acid sequence Colicin A was purified as previously described (Cavard and Lazdunski, 1979). The sequence of the N-terminal 12 amino acids of the protein was determined by automated Edman degradation in a Beckman sequenator. Phenylthiohydantoin derivatives of amino acids were identified by h.p.l.c.
Acknowledgements We are very grateful to Dr.P.Cossart for introducing one of us to 'the ins and outs' of DNA sequencing and to Drs.D.Carvard and S.Varenne for fruitful discussions. This work has been supported by a grant from the ATP 'Biologie Moleculaire du Gene' and the 'Fondation pour la Recherche Medicale'.
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Howard-Flanders,P. and Boyce,R.P. (1966) Radiat. Res. Suppl., 6, 156-184. Kenyon,C. and Walter,G. (1981) Nature, 289, 805-810. Kirby,E.P., Jacob,F. and Goldthwait,D.A. (1967) Proc. Natl. Acad. Sci. USA, 58, 1903-1910. Little,J.W. and Mount,D.W. (1982) Cell, 29, 11-22. Maxam,A. and Gilbert,W. (1980) Methods Enzymol., 65, 499-560. Miki,T., Ebina,Y., Kishi,F. and Nakazawa,A. (1981) Nucleic Acids Res., 9, 529-543. Miura,A. and Tomizawa,J. (1968) Mol. Gen. Genet., 103, 1-10. Morlon,J., Cavard,D. and Lazdunski,C. (1982a) Gene, 17, 317-321. Morlon,J., Cavard,D. and Lazdunski,C. (1982b) FEBS Lett., 141, 116-119. Pribnow,D. (1975) Proc. Natl. Acad. Sci. USA, 72, 784788. Rosenberg,M. and Court,D. (1979) Annu. Rev. Genet., 19, 319-353. Sancar,G.B., Sancar,A., Little,J.W. and Rupp,W.D. (1982) Cell, 28, 523530. Shine,J. and Dalgarno,L. (1974) Proc. Natl. Acad. Sci. USA, 71, 1342-1346. Tomizawa,J. and Ogawa,J. (1968) Cold Spring Harbor Symp. Quant. Biol., 3, 248-251. Varenne,S., Cavard,D. and Lazdunski,C. (1981) Eur. J. Biochem., 116, 615-620. Waller,J.P. (1963) J. Mol. Biol., 7, 483-496. Yamada,M., Ebina,Y., Miyata,T., Nakazawa,T. and Nakazawa,A. (1982) Proc. Natl. Acad. Sci. USA, 79, 2827-2831.
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