LexA Protein Is a Repressor of the Colicin E l Gene

‘lHE .JOURNAL OP RIOLOGICAI. CHEMISTRY Vol. 258, No. 21, Issue of November IO, pp. 1325&1926l, 198:i I’nntrd In (T..S A

LexA Protein Is a Repressor of the Colicin E l Gene* (Received for publication, March 4, 1983)

Yousuke Ebina, Yoshiyuki Takahara, Fumio Kishi, and Atsushi NakazawaJ: From the Department of Biochemistry, Yamaguchi University School of Medicine, Ube, Yamaguchi 755, Japan

Roger Brent$ From the Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138

EXPERIMENTALPROCEDURES

Purification ofLexA Protein-LexA protein was purifiedfrom RB791 cells harboring pRB451, a plasmid carrying a fusion between the lexA gene and a modified lac promoter that directs the synthesis of large amounts of LexA protein.’ Purification procedures were as described by Brent and Ptashne(8), except that Sephadex G-100 was used instead of Bio-Gel P-150 and the final step of the organomercurial-agarose column was omitted.Protein was mesured by the method of Lowry et al. (10) with bovine serum albumin asa standard. Molarities of LexA protein were calculated as concentrations of a monomer, the molecular weight of which is 22,700 (11). The LexA protein thus obtained hada physical purity of at least 95% as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We confirmed that the proteinwas cleaved by RecA (ti7) protease (4) and specifically inhibited the synthesis of RecA and LexA mRNAs in vitro (7, 8). In Vitro Transcription-Transcription was performed as described (6) except that templates were at 33 nM. Incubations were carried out Colicin E l is an antibiotic protein encoded by a plasmid, in two stages. (i) LexA protein was added to the reaction mixtures ColE1. The gene for colicin E l is dormant under ordinary for the transcription, from which RNA polymerase was omitted (ii) conditions but becomes active with regard to colicin E l pro- after leaving the mixtures for 10 min a t 37 “C to allow LexA protein duction on treatments that damageDNA or inhibit DNA binding to the operator, RNA polymerase was added to initiate the replication. The colicin E l induction is considered to be an transcription. After 15 min, the reaction was terminated by shaking with phenol. Transcripts were analyzed on a 6 or 8%polyacrylamide, “SOS reponse,” which includes phenomena such as enhanced 8 M urea gel and visualized autoradiographically. For transcription DNA repair capacity, induced mutagenesis, inhibition of cell analysis of the colicin E l gene, the 571-bp2 SmaI-HaeIII fragment division (filamentation),cessation of respiration,andprocontaining the colicin El regulatory region (Fig. 1) was used as a phage induction (1, 2). The SOS response is controlled co- template. Thecolicin E l mRNA transcriptswere labeled with [y-”PI of the RecA mRNA ordinately by two regulatory elements, RecA and LexA pro- GTP aspreviously described (6). For the analysis teins. LexA protein appears to operate exclusively as a re- synthesis, the302-bp HaeIII fragment containing therecA regulatory region was used (12). The RecA mRNA and RNA-1 of pA03 (13) pressor of “SOS genes” (3). RecA protein, which is activated were labeled with [y-32P]ATP. DNA was measured by fluorometric as a protease by signals occurring after SOS-inducing treat- assay (14). ments (4), inactivates LexA protein ( 5 ) . Thus, theSOS genes DNase I Protection Experiments and Binding Affinity-The experiments of DNase I protection(“footprinting”),todeterminethe were derepressed to elicit SOS responses. We reported that the operator-promoterregion of the coli- stretch of DNA covered by LexA protein and to estimate thebinding cin E l gene has DNA sequence homology to the operators of affinity of LexA protein to the colicin E l operator, were done by using the procedure of Johnson et al. (15). Thereaction mixture (200 the recA and lexA genes (6), which are commonly repressed SI) containing 10 mM Tris-HCI, pH 7.0, 2.5 mM MgCla, 1 mM CaCl,, by LexA protein (7, 8). These findingsplus the results of 0.1 mM EDTA, 200 mM KCI, 100 Fg/ml of bovine serum albumin, 2.5 analysis using a double mutant of the recA and lexA genes (9) rg/ml of salmon sperm DNA, labeled DNA, and various amounts of led to the hypothesis that LexA protein directly represses the LexA protein was preincubated for 15 min a t 37 “C. After adding 2 was colicin E l gene. We now present evidence that LexA protein ng/ml of pancreaticDNase I to the reaction, the incubation is indeed a repressor of the colicin E1 gene. The mode of LexA continued for 10 min at 37 “C. The reaction was stopped with 50 ~1 of cold 8 M ammonium acetate and tRNA a t 300 pg/ml. The DNA protein binding to the colicin E l operator region is also was precipitated with ethanol and analyzedby electrophoresis on an described. 8% polyacrylamide, 7 M urea gel, followed by autoradiography. The 154-bp Sau3A-SstIIand 153-bp ThaI-Sau3Afragments of ColEl containing the operator region (Ref. 6; see Fig. 1) were labeled with ”’P at the 5’ ends of Sau3A and ThaI sites, respectively, and then * T h i s work was supported by Grants-in-Aid forScientific Re- subjected to the DNaseI protection experiments. The affinity of the colicin E l operator for LexA protein was determined according to the search from the Ministryof Education, Science, and Cultureof Japan. The costs of publication of this article were defrayed in part by the principle described by Riggs et al. (16). ”P-labeled restriction fragments added were such that the operator concentration was 50.05 payment of page charges. Thisarticlemusttherefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 nM. A t this concentration, the apparentKd is approximatelyequal to the concentrationof LexA protein required to occupy one-half of the solely to indicate this fact. $ To whom correspondence should be addressed. ’ R. Brent, unpublished observations. §Supported by Grant GM29988 from the National Institutes of The abbreviation used is: bp, base pair. Health. ~~

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LexA protein is a represssor of several chromosomal genes involvedin the SOS response in Escherichia coli. In previous experiments, we found that LexA protein may also be a repressor of the colicin E l gene. We now present evidence that the purified LexA protein strongly repressed the in vitro transcription of the colicin E l gene. As determined in DNase I protection experiments, LexA protein bound with a high affinity to the approximately 40-base pair long sequence betweenthe Pribnow boxand the start codon of the colicin E l gene. The sequence of the binding site was composed of two overlapped “SOS boxes” to which the LexA protein bound in a cooperative manner.

LexA Repressor of Colicin E l Gene

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mRNA-I mRNA-2

LexA binding region 0 1

-300

1

I

-200 1 -100

I

mRNA-I+ .L base pairs

I

100

200

300

FIG. 1. The operator-promoter region of the colicin E l gene. The structural gene for colicin E l is shown by the stippled box. The positions of the restriction enzyme sites used in this study are shown by vertical arrows. Two in vitro transcripts of the colicin E l gene are shown by wavy lines. Only mRNA-1 is synthesized in vivo (18). LexA protein binding site determined in the present study is indicated by a dark box. Distances are given in base pairs from the initiation site of mRNA-1.

RESULTS ANDDISCUSSION

Specific Inhibition of in Vitro Transcription-From in vitro transcriptionexperiments using the 571-bp SmI-HaeIII DNA fragment containing the promoter-operator region of the colicin E l gene, we previously showed that two species of RNA, 289 nucleotides (mRNA-1) and 205 nucleotides (mRNA-2), were synthesized (6). The mRNA-1 and mRNA2 started, respectively, 75 bp upstream and 10 bp downstream from the NH2-terminal codon of the colicin E l gene. Although synthesis of mRNA-2 was four times as efficient as that of mRNA-1 in vitro (Fig. 2a), we identified by S1-mapping assay the promoter of mRNA-1 as the authentic promoter of colicin E l mRNA synthesis in vivo (18). The physiological role of the promoter of mRNA-2 is unknown. As shown in Fig. 2a, LexA protein strongly inhibited the synthesis of mRNA-1, but weakly that of mRNA-2. In thecontrol, RNA-1 synthesis of pA03 (13) was not inhibited by the protein (Fig. 2c). These results indicate that in vitro synthesis of colicin E l mRNA is specifically inhibited by LexA protein. Although it was suggested that a peculiar repressor protein of the colicin E l gene might be encoded by the plasmid (19), we obtained evidence that such a repressor protein is not synthesized from the plasmid.3Therefore, LexA protein is considered to be a proper repressor of the colicin E l gene. The genes, which LexA protein has so far been reported to repress, were the chromosomal genes such as recA (7, 8), l e d (7, 8), uvrA (20), uvrB (21), SUM: d i d , and dinB (22). The above findings indicate that the extrachromosomal gene is also subject to repression byLexA protein. It was suggested that LexA protein is also a repressor of the colicin E2 (ll),E3,5 cloacin DF13 (23), and pKMlOl rnuc gene (24). As shown in Fig. 2a, the in vitro synthesis of colicin E l mRNA was completely inhibited a t 100 nM LexA protein.

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Y. Ebina, Y. Takahara, K. Shirabe, M. Yamada, T. Nakazawa, and A. Nakazawa, manuscript in preparation. ‘S.Mizusawa, presentated a t the5th Annual Meeting of the Molecular Biology Society of Japan, December 9,1982, Tokyo, Japan. H. Masaki, presentated at the 5thAnnual Meeting of the Molecular Biology Society of Japan, December 10, 1982,Tokyo, Japan.

0 +

RecA mRNA

*RNA-1

FIG. 2. Inhibition of colicin E l transcription by LexA protein in vitro. mRNA-1 (289 nucleotide) and mRNA-2 (205 nucleotide) in a, RecA mRNA (96 nucleotide) in b, and RNA-1 (110 nucleotide) in c were analyzed on polyacrylamide-urea gels. The experimental protocol is described under “Experimental Procedures.” Lanes 1,2, and 3 are 0, 100, and 200 nM LexA protein added to the reaction mixture, respectively. Faint bands just above rnRNA-I and below rnRNA-2 in lanes 2 and 3 of a were probably due to the transcripts which started a t both ends of the template DNA and stopped at theLexA protein binding region.

However, at the same concentration of the protein, the in vitro synthesis of RecAmRNA decreased to 60% of the control (Fig. 2b). L e d Protein Binding Site-To assess the mechanism of the strongrepression of the colicin E l transcription by LexA protein, we determined the binding site of the protein to the control region of the gene. From the footprinting experiments using both of the strands,we found that LexA protein bound to a segment of DNA downstream from the Pribnow box (Fig. 3, a and b). The segment lies about 10 bp upstream to 30 bp downstream from the start site of the colicin E l mRNA synthesis. It overlies a 37-bp inverted repeat that we previously speculated to be the binding site of LexA protein to the colicin E l gene (6). The protected region of the colicin E l gene seems to be composed of two overlapped “SOS boxes” (7). Strong homology is observed in the sequence of the left binding site, whereas the sequence of the right binding site is less homologous, as shown in Fig. 4. Affinity of Binding-The equilibrium dissociation constant for LexA protein to thecolicin E l control region was measured under the conditions (37 “C,200 mM KCI) where Kd values of LexA protein for the recA and l e d operators were determined (8). A t an operator concentration (50.05 nM), which is far below the dissociation constant for LexA protein to thebinding site, the Kd should be approximately equal to theconcentration of LexA protein required to occupy one-half of the operator molecules (16). As shown in Fig. 3a, about 70% of the operator molecules were occupied by LexA protein a t 0.5 nM, while all of them were bound by the protein at 1.0 nM. Therefore, the apparentKd of LexA protein for the colicin E l operator was about 0.4nM. However, the actual Kd may be lower than this value, because some of the -in may be incompetent to bind the operator a t these -&Ions. The apparentKd value of LexA protein for the recA operator, which was determined under the same conditions, wasre-


operator molecules (8).The intensity of the protected band in footprinting was determined by inspection. Materials-Restriction endonucleases and T, polynucleotide kinase were purchased from Takara Shuzo (Kyoto, Japan). DNase I was obtained from Boehringer Mannheim. [Y-~’P]ATP(5400 Ci/ mmol) and [y-”P]CTP (36 Ci/mmol) were products of the Radiochemical Centre (Amersham). RNA polymerase was purified by a modification of the method of Chamberlin and Berg (17).

mRNA-2-

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LexA Repressor of Colicin E l Gene

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+X

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10

a-

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consensus

~TGTATATNNANNCA~ FIG. 4. Location and consensus sequence of LexA protein binding sites in the promoter-operator regions of various genes that are regulated by LexA protein. Pribnow boxes are underlined. Numbering is from the transcription start site of each mRNA. The regions protected from DNase I digestion byLexA protein are bracketed. Presumed SOS boxes are indicated by boxes. The postulated sites for LexA protein binding in SUM and cloacin DF13 genes are presented. In the SOS boxes, highly preserved positions are indicated by large capital letters; other preserved positions are indicated by small capital letters.

ported to be about 2 nM (8).Although a comparison of the values from differentexperiments may not be valid, the affinity of LexA protein for the colicin E l operator appears higher than that for the recA operator. A t 0.25nM, LexA protein did not occupy the colicin E l operator, but at 0.5 nM the protein simultaneously bound to both of the SOS boxes. Binding of LexA proteintothe

Acknowledgments-We thank Drs. K. Tomita and T. Nakazawa for helpful discussion and M. Ohara for reading the manuscript. REFERENCES 1. Radman, M. (1975) in Molecular Mechanisms for Repair of DNA (Hanawalt, P. C., and Setlow, R. B., eds) pp. 355-367, Plenum Publishing Corp., New York 2. Witkin E. W. (1976) Bacteriol. Rev. 40,869-907 3. Little, J. W., and Mount, D. W. (1982) Cell 29, 11-22


FIG. 3. Binding of LexA protein to the colicin E l operator. Footprinting was done as described under "Experimental Procedures." a, about 0.05 nM 154-bp Sau3A-SstII fragment of ColEl containing the operator region which was labeled with 32Pat the 5' end of Sau3A was used (antisense strand). The chemical sequencing was done by the method previously described (6). b, about 0.5 nM 153-bp ThaI-Sau3A fragment labeled with 32Pat the5' end of ThaI was used (sense strand) to demonstrate the LexA protein binding region. The bases were numbered relative to the starting point of colicin E l mRNA, which was designated + I . Brackets indicate portions of the region that were protected by LexA protein from digestion with DNase I. The lower site of the region protected by LexA protein in b was visualized more clearly by a longer exposure (not shown).

operator did not show a linear relationship between LexA protein and the operator occupied by the protein. What is observed is consistent with the idea that LexA protein binds to thecolicin E l operator in a cooperative manner. Consensus Sequence of L e d Protein Binding Sites-In Fig. 4, the LexA protein binding sequence of the colicin E l gene is compared with operators of l e d (7, 8), recA (7, 8), uurA (20), and uurB (21) genes and also with presumed operators of SUM (25) and cloacin DF13 (23) genes. The structure of thesebindingsites (SOS box) has a consensus sequence, "'CTGTATATNNANNCAGI'. The3-bpsegments at the ends of this 16-bp sequence are related by a 2-fold axis of symmetry. The terminal 3-bp segments are thought to be importantto LexA protein binding from the methylation experiments of the recA and l e d genes carried out by Brent and Ptashne (8).Some of the repressor proteins so far characterized exist ina dimeric form which has a %fold symmetry axis coincident with the 2-fold axis in thebinding site of the DNA molecule (26,27). SinceLexA protein tends todimerize (8), the protein that undergoes interaction with the above binding site would probably also be in a dimeric form. The classical B-form DNA has 10.0 bp/turn of the helix. Therefore, the terminal segmentsin each SOS box separated by 10 bp eventually face to nearly opposite directions on the sides of the DNA molecule. LexA protein would interact with the terminal 3-bp segments which are exposed within the major groove of the DNA as predicted in the case of other repressor proteins (26, 27). On the other hand, the D-form DNA which has 8.0 bp/turn reportedly exists in the DNA with a high content of A-T (28, 29). In this form of DNA, it is also possible to assume that the protein can interactwith the 3-bp segments along one face of the DNA molecule. The sequence of A-T was frequently observed in these SOS boxes. In the B-form DNA, an axial rise/residue is 0.34 nm, while it is 0.3 nm in the D-form DNA (28, 29). Thus, the length of the SOS box should be 5.44 nm in the B-form DNA and 4.8 nm in the D-form DNA. Assuming that a dimer molecule of LexA protein is a sphere, the diameter would be 5.7 nm. Therefore, the LexA dimer could adequately cover the 16-bp sequence of either the B- or the D-form of DNA. Another feature of the LexA binding site of the colicin E l operator is that there are two overlapping SOS boxes. The structure of the rightbinding site is less homologous with the typical SOS box. However, the LexA protein binding to both boxes occurred at the same concentration of the protein (Fig. 2a). This hypothetical cooperative binding could occur by at least two mechanisms. (i) Binding of LexA protein to one of the SOS boxes would induce a conformational change in the neighboring box and lead to stimulation of another molecule of LexA protein tobind to thebox; ii) LexA protein bound to one of the SOS boxes could interact with another molecule with protein-protein association and stimulate theLexA protein binding to the adjacent box. LexA protein binds with high affinity to the colicin E l operator site which is located in the3' region of the Pribnow box. This high affinity binding probably accountsforthestrong repression of colicin E l mRNA synthesis by LexA protein.

LexA Repressor of Colicin E l Gene 4. Craig, N. L., and Roberts, J. W. (1980) Nature (Lond.)283, 2630 5. Little, J. W., Edmiston, S. H., Pacelli, L. Z., and Mount, D. W. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 3225-3229 6. Ebina, Y., Kishi, F., Miki, T., Kagamiyama, H.,, Nakazawa, T., and Nakazawa, A. (1981) Gene (Amst.) 15,119-126 7. Little, J. W., Mount, D. W., and Yanisch-Perron, C. R. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4199-4203 8. Brent, R., and Ptashne, M. (1981) Proc. Natl. Acad. Sei. U. S. A. 78, 4204-4208 9. Ebina, Y.,Kishi, F., and Nakazawa, A. (1982) J . Bacteriol. 150, 1479-1481 10. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J . Biol. Chem. 193,265-275 11. Horii, T., Ogawa, T., and Ogawa, H. (1981) Cell 23, 689-697 12. Horii, T., Ogawa, T., and Ogawa, H. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 313-317 13. Morita, M., and Oka, A. (1979) Eur. J. Biochem. 97,435-443 14. Hinegardner, P. T. (1971) Anal. Biochem. 39, 197-201 15. Johnson, A. D., Meyer, B. J., and Ptashne, M. (1979) Proc. Natl. Acad. Sci. U, S. A. 76,5061-5065 16. Riggs, A. D., Suzuki, H., and Bourgeois, S. (1970) J. Mol. Biol. 48,67783 17. Chamberlin, M., and Berg, P. (1962) Proc. Natl. Acad. Sci. U. S.

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