Proc. Natl. Acad. Sci. USA Vol. 90, pp. 2030-2034, March 1993 Biochemistry Orientation of the LexA DNA-binding motif on operator DNA as inferred from cysteine-mediated phenyl azide crosslinking (SOS response/gene regulatlon/LexA repressor/DNA recognition/4-azidophenacyl bromide) PASCAL DUMOULIN, PASCALE OERTEL-BUCHHEIT, MICHtLE GRANGER-SCHNARR, AND MANFRED SCHNARRt Institut de Biologie Moleculaire et Cellulaire, UPR 9005 du Centre National de la Recherche Scientifique, 15, rue Ren6 Descartes, F-67084 Strasbourg, France Communicated by Jean-Marie Lehn, November 5, 1992 (received for review June 30, 1992)

ABSTRACT To address the question how the recognition residue in position 52, close to the COOH terminus of helix helix of the LexA repressor is positioned within the major 3. Site-specific incorporation of small bifunctional photo- groove of operator DNA we have applied a site-specific pho- crosslinking or DNA-cleaving agents allows mapping of the tocrosslinking approach using a LexA mutant repressor (LexA- position of an amino acid relative to the DNA bases within a C52) that harbors a single cysteine side chain in position 52, protein-DNA complex, as shown earlier with EDTA'Fe (16- close to the COOH terminus of helix 3. The LexA-C52 mutant 20) or phenanthroline adducts (21). Site-specific incorpo- repressor has been purified and modified site-specifically with ration ofphotoactivatable crosslinking agents such as phenyl the photoreactive azido compound 4-azidophenacyl bromide, azide has been successfully used in the study of DNA giving rise to LexA-CS2*. Here we show that LexA-C52* may triple-helix formation (22, 23) and more recently in the be selectively photocrosslinked with two adjacent bases within establishment of protein-DNA contacts in the case of the each operator half-site. The crosslinked bases are located, catabolite gene activator protein (24). Here we use the respectively, 10 and 11 base pairs from the dyad axis of the bifunctional photoactivatable reagent 4-azidophenacyl bro- operator. The crosslinking data imply that the LexA recogni- mide to show that helix 3 of LexA may be crosslinked with tion helix is oriented opposite to what is generally observed for operator DNA. helix-turn-helix proteins and that this helix should form a steeper angle with respect to the plane of the base pairs than is MATERIALS AND METHODS helix-turn-helix proteins. observed for standard Protein Purification. The mutant repressor LexA-C52 (i.e., Arg-52 -- Cys) was purified essentially as described (7) The LexA repressor from Escherichia coli regulates the except that the protein eluted from the phosphocellulose transcription of about 20 different so-called SOS genes, column at about 80 mM NaCl instead of 280 mM in the case which are mostly involved in DNA repair, mutagenesis, DNA of the LexA wild-type protein. Accordingly, the ionic replication, and (for reviews see refs. 1-4). LexA strength of the loading buffer was reduced to 40 mM NaCl. is a two-domain protein of 202 amino acids that binds DNA The protein was stored at -80°C in 200 mM KCI/20 mM via its NH2-terminal domain (5, 6). The protein dimerizes by Tris'HCl, pH 7.3/5 mM 2-mercaptoethanol/5% (vol/vol) means of its COOH-terminal domain with a fairly small glycerol. dimerization constant of 2 x 104 M-1 (7, 8) and regulates As judged from gel retardation experiments, LexA-C52 transcription upon binding to its palindromic operator se- binds to a 50-bp SOS consensus operatorfragment with about quences such that two LexA monomers bind sequentially and one-fourth to one-third the affinity ofLexA wild-type repres- cooperatively to the two operator half-sites (9). The NH2- sor. The sequence of the synthetic operator duplex used for terminal DNA-binding domain of LexA shows only weak most of the experiments was sequence homology with known DNA-binding motifs, in- cluding helix-turn-helix (HTH) motif proteins (4, 10). A S'-GACGATGGCACCCTAZACTGTATATATATICAGTAATCCCGTAGCGGCGG structural NMR investigation showed that the conformation 3'-CTGCTACCGTGGGAZ.TG&CITATATATATGTCATTAGGGCATCGCCGCC, ofthe NH2-terminal domain is not more closely related to the where the two CTGT recognition motifs are shown in bold- class of HTH proteins than to proteins without known face letters. Some experiments were done with a slightly DNA-binding activity (11). At present only low-resolution modified version of this duplex for which the two underlined NMR data on the LexA-DNA complex are available (12). base pairs were replaced by a GC CG sequence to assess ifall Sequence conservation among the SOS operators suggests four nucleotides may be crosslinked in these positions. that an optimal operator should have a twofold symmetric Protein Modification with 4-Azidophenacyl Bromide. LexA- CTGT(AT)4ACAG sequence (13), and indeed this sequence C52 (0.4 ml, 50 uM) was incubated for 3 hr with 3.5 mM binds LexA more tightly than any naturally occurring SOS 4-azidophenacyl bromide (Sigma) at 23°C in a buffer contain- operator (4). The major recognition elements should be ing 100 mM KCl, 20 mM Mops at pH 8.0, 1.25% (vol/vol) comprised in the two outer elements (the CTGT motifs) as glycerol, and 1.25 mM 2-mercaptoethanol, giving rise to judged from the distribution of operator-down mutations LexA-C52*. Unreacted 4-azidophenacyl bromide was re- (compiled in ref. 4), chemical protection and interference moved by chromatography on a Bio-Gel P-6DG (Bio-Rad) data (14), and NMR results (12). column (1 x 27 cm). A random mutagenesis study of the LexA protein sug- The degree of modification with phenyl azide was deter- gested that helix 3 of LexA (aa 41-54) should be involved in mined by measuring the amount of carboxymethylcysteine DNA recognition (15). To test this hypothesis further, we upon reaction with iodoacetic acid. The relative amount of have purified and chemically modified a LexA mutant protein carboxymethylcysteine with respect to the serine peak was 0 containing a single cysteine residue instead of an arginine for LexA wild-type repressor containing no cysteine resi-

The publication costs ofthis article were defrayed in part by page charge Abbreviations: HTH, helix-turn-helix; LexA-C52*, LexA mutant payment. This article must therefore be hereby marked "advertisement" (Arg-52 Cys) repressor modified with 4-azidophenacyl bromide. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed.

2030 Downloaded by guest on September 25, 2021 Biochemistry: Dumoulin et al. Proc. Natl. Acad. Sci. USA 90 (1993) 2031 dues, 0.98 for LexA-C52 containing a single cysteine, and 0 SDS/polyacrylamide gel electrophoresis (Fig. 1). The again for LexA-C52 after modification with 4-azidophenacyl amount of crosslinked DNA after 5 min of UV irradiation is bromide, suggesting that essentially all the cysteine side about 2-3%, as judged from densitometry of the autoradio- chains have reacted with 4-azidophenacyl bromide and are no graphs. Further irradiation does not increase the amount of longer free to interact with iodoacetic acid. crosslinked DNA, probably due to the photodegradation of Protein-DNA Photocrosslinking. LexA-C52* (3.2 ,uM) was the phenyl azide moiety. incubated at 20°C for 30 min in a buffer containing 20 mM To map the positions of the nucleotides which have been Mops at pH 7.3 and 100 mM KCl with an operator DNA crosslinked to this LexA derivative, the crosslinked DNA has fragment (50-mer), which was 32P-labeled at the 5' end of been subjected to cleavage with piperidine (25). Treatment of either the top or the bottom strand. Aliquots (50 .ul) were then the crosslinked protein-DNA complex with piperidine at high withdrawn and irradiated with a 6-W Vilber Lourmat VL- temperature results in DNA cleavage at the crosslinked 6MC UV lamp (312 nm) at a distance of about 20 cm for 1, 3, nucleotides. Fig. 2 shows that crosslinking on the top strand or 10 min. The samples were further subjected to three takes place preferentially at two adjacent nucleotides close to extractions with phenol in which the aqueous phase was followed a with discarded, by precipitation ethanol and LexA-C52* cleavage with piperidine (1 M) for 30 min at 90°C. No DNA g sEiE *e cleavage products were observed without piperidine treat- ment, showing that phenyl azide crosslinking alone does not give rise to strand cleavage. Chemical G>A and G+A sequencing reactions were as described (25). High-resolution gel electrophoresis was carried out in 20% polyacrylamide denaturing gels.

RESULTS A~ ~~~~ In this study we use the photoactivatable crosslinking agent 4-azidophenacyl bromide to map LexA-DNA contacts. Since the wild-type LexA repressor does not contain any *1. cysteine side chains, a mutant repressor had to be used for this study. In an earlier genetic study (15) we have identified a suitable candidate, LexA-C52, which harbors an Arg-* Cys mutation in position 52. Gel retardation experiments with the purified LexA-C52 repressor showed that this LexA variant A olo binds to the consensus SOS operator with about one-fourth T to one-third the affinity ofthe LexA wild-type repressor (data not shown). )P A The purified LexA-C52 protein was modified with the TtR cysteine-specific bifunctional photoactivatable crosslinking :: agent 4-azidophenacyl bromide under conditions in which essentially all the cysteine-52 side chains were modified, giving rise to a protein species that we will call in the T following LexA-C52*. To assess protein-DNA crosslinking with this LexA derivative we have incubated LexA-C52* IC with a 32P-labeled duplex DNA harboring the consensus SOS operator sequence, irradiated the reaction mixtures with 312-nm UV light, and analyzed the reaction products by

c C 146mmim, lqw: - crosslinked LexA-C52*/DNA .._.. . complex

- DNA

FIG. 2. Autoradiograph of a high-resolution denaturing poly- FIG. 1. Autoradiograph of a denaturing (SDS) polyacrylamide acrylamide gel revealing the site-specific crosslinking of LexA-C52* gel, showing the formation, after UV irradiation for the indicated to the top strand of a synthetic 50-bp DNA duplex containing the times, of a slowly migrating crosslinked complex between the consensus SOS operator sequence. Two arrows indicate specific purified LexA-C52 mutant repressor harboring a phenyl azide group piperidine cleavage products for an adenine and a thymine base in position 52 (LexA-C52*) and a 50-bp DNA duplex harboring the situated, respectively, 3 and 2 bases 5' to the highly conserved CTGT consensus operator sequence. motif. Downloaded by guest on September 25, 2021 2032 Biochemistry: Dumoulin et al. Proc. Natl. Acad Sci. USA 90 (1993) the CTGT motif. The two symmetrically related nucleotides LexA wild type (M) 4~ on the bottom strand are crosslinked with the same efficiency and specificity as those on the top strand (data not shown). ol. 0. Within both strands the crosslinked nucleotides are situated, respectively, 2 and 3 base pairs 5' to the first nucleotide ofthe highly conserved CTGT motifs. Crosslinking occurs with two T adjacent A and T nucleotides on the top strand and two adjacent T nucleotides on the bottom strand. A A a For four crosslinked we U. of all nucleotides observe two pi- .W,:" -- G "*-: peridine cleavage products with about equal intensity, one of which comigrates with the standard sequencing products T whereas the other shows a slightly reduced electrophoretic C C mobility. It seems likely that the second species contains the same number of negatively charged phosphate groups but C C some additional mass, for example an additional deoxyribose FIG. 3. (Left) Autoradiograph of a high-resolution polyacrylam- moiety, leading to a slightly reduced electrophoretic mobil- ide gel showing that increasing amounts of unmodified LexA wild- ity. Further work is necessary to elucidate the nature of the type repressor suppress photocrosslinking of LexA-C52* to the top second species. The results obtained so far indicate that this strand of the same DNA duplex as that used in Fig. 2. (Right) species appears also when the crosslinked DNA is cleaved Autoradiograph ofa high-resolution polyacrylamide gel showing that with NaOH (0.1 M, 30 min at 90°C) or with a piperidine a 50-bp duplex harboring a GC sequence instead of AT in positions concentration of2 M instead of 1 M, or when photocrosslink- -11 and -10 results in the same crosslinking pattern as the AT- ing is induced with UV light ofa different wavelength (254 nm containing duplex. or 360 nm instead of 312 nm). tion helix should be oriented with respect to the operator To make sure that crosslinking is due to the formation of sequence. Earlier genetic data have pointed to helix 3 as the a specific LexA*DNA complex, two control experiments major element for operator DNA recognition (15). The have been performed: First, no crosslinking was observed crosslinking data reported here imply that this helix must with a DNA fragment lacking the specific operator DNA site indeed be in close contact with the DNA, that it must be for LexA binding (data not shown). Second, photocrosslink- positioned such that its COOH terminus is rather far from the ing of LexA-C52* is suppressed upon addition of increasing dyad axis of the operator, and that recognition occurs by amounts ofunmodified LexA repressor (Fig. 3 Left). Several means of the NH2-terminal part of helix 3 and possibly by other control experiments (see Fig. 2) have been done, means of the turn between helix 3 and helix 2. showing that crosslinking occurs only for LexA-C52*, not for Fig. 4 also shows those phosphate groups for which LexA-C52 and LexA wild-type repressor. ethylation suppresses complex formation with the LexA A crosslinking agent might have some selectivity with wild-type repressor (26). Since one ofthese phosphate groups respect to the target constituents. The experiments outlined is foundjust between the two crosslinked nucleotides on both above have shown that LexA-C52* may be crosslinked to DNA strands, we wondered if the positively charged Arg-52 both A and T. To assess if G and C may also be crosslinked side chain could be responsible for this interference. Exper- in our system, we have replaced the reactive A and T on the iments similar to those described in ref. 26 showed, however, top strand by G and C. Fig. 3 Right shows that G and C are that the LexA-C52 mutant repressor has the same ethylation indeed crosslinked in the same position and with comparable interference pattern as the LexA wild-type repressor (data efficiency. We may conclude from these results that all four not shown). nucleotides may be efficiently photocrosslinked. Fig. 4 summarizes the crosslinking contacts between the DISCUSSION SOS consensus operator and the LexA derivative used in this We show that a derivatized amino acid side chain situated study and suggests further how the putative LexA recogni- close to the COOH terminus ofhelix 3 ofthe LexA repressor

-12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 T rA'Wf-A-C-T *G T A T A T A-T *A T AC T A1A T A TA T GKA 7 T ATA T A T A AOl A 1 3~~~~~~~~~ ~~~~~~~~~~12 3 4 5 6 7 8 9 10 11 12

FIG. 4. Diagram showing a juxtaposition of helix 3 of LexA and the SOS consensus operator sequence with a numbering system relative to the symmetry axis of the operator (*). The two CTGT recognition motifs are boxed and the crosslinking contacts are indicated by arrows. Amino acids are written in the one-letter code. The black dots indicate those phosphate groups for which ethylation suppresses complex formation (26). Downloaded by guest on September 25, 2021 Biochemistry: Dumoulin et al. Proc. Natl. Acad. Sci. USA 90 (1993) 2033 can be specifically crosslinked to nucleotides which are a first step a physical model (using Maruzen molecular located close to the CTGT recognition motif of an SOS models) ofthe LexA-DNA complex, using a standard B-DNA consensus operator. As inferred from methylation protection duplex. Docking of the protein was achieved such that the and interference experiments (6, 14, 27), LexA binds DNA exocyclic azide moiety of the phenyl ring attached to Cys-52 predominantly within the major groove. Placing the COOH was positioned in the major groove of the DNA such that it terminus of helix 3 within the major groove of standard may interact with the two crosslinked nucleobases. Glu-45 B-DNA, such that the phenyl azide moiety in position 52 may was postulated to form a hydrogen bond with the exocyclic contact the crosslinked nucleotides, very naturally brings the NH2 group ofthe cytosine base within the CTGT motif (base NH2 terminus ofhelix 3 close to the CTGT recognition motif pairs ± 8 in the numbering system proposed in Fig. 4). Given (Fig. 5). This finding is in good agreement with a recently the crosslinking data, docking of Glu-45 to the opposite discovered LexA mutant repressor with a different DNA guanine would lead to serious clashes between the NH2 recognition specificity (29), suggesting that the side chain of terminus of helix 3 and the DNA backbone. In a second step Glu-45 should recognize preferentially the C-G base pair in the physical model was reproduced on a Silicon Graphics the first position of the CTGT recognition motif. workstation. Crosslinking and genetic data together allow us to realize The result ofthis docking procedure is shown in Fig. 5. The a fairly precise docking ofhelix 3 into the major groove ofthe left side shows our model for the complex between the LexA DNA and to compare its orientation with that of the recog- recognition helix and one operator half-site. The right side nition helix of a standard HTH protein. On the basis of the shows part of the cocrystal structure of the phage A structure of the LexA DNA-binding domain we have built in repressorDNA complex (28). In both cases the protein

FIG. 5. (Left) An a-helix having the sequence ofamino acids 39-54 ofthe LexA repressor (SPNAAEEHLKALARKG, residues 39-54) has been docked to a dodecamer of standard B-DNA (corresponding to the right half-operator shown in Fig. 4-i.e., base pairs 1-12) generated with INSIGHT ii. According to recent NMR results helix 3 of LexA is essentially straight (R. Fogh, R. Kaptein, H. Rfiterjans, M.G.-S., and M.S., unpublished work). The four base pairs of the CTGT motif are situated in the center ofthe dodecamer. The phenyl azide moiety (shown as the reactive nitrene intermediate formed after N2 release upon UV irradiation) attached to the cysteine in position 52 (labeled as Cys) and the side chains of Glu-45, Asn-41, and Ser-39 are shown in yellow. To realize this docking the phenyl azide ring was placed into the major groove at about equal distance from the two crosslinked bases (labeled with red arrows) and Glu-45 was positioned close to the exocycic NH2 group of the cytosine base (labeled with a white arrow) of base pair 8. (Right) Part of the crystal structure of the complex of the phage A repressor with its DNA- (28). Only the recognition helix and two amino acids of the preceding and two amino acids of the following turn are represented (residues 42-54: MGQSGVGALFNGI). The first amino acid ofthe recognition helix (Gin) is labeled and, as in the case ofthe model of the LexA-DNA complex, the dyad axis of the operator is placed at the bottom of the figure. Downloaded by guest on September 25, 2021 2034 Biochemistry: Dumoulin et al. Proc. Natl. Acad. Sci. USA 90 (1993) segments (shown as purple ribbons) correspond to the rec- Recherche M6dicale (871007), the Association de Recherche contre ognition helix and the two NH2-terminal amino acids of the le Cancer, and the Ligue Nationale de la Lutte contre le Cancer. preceding turn. Fig. 5 shows that the positioning ofthe LexA 1. Little, J. W. & Mount, D. W. (1982) Cell 29, 11-22. recognition helix should be fairly different from that observed 2. Walker, G. C. (1984) Microbiol. Rev. 48, 60-93. for the phage A repressor recognition helix. The most obvious 3. Little, J. W. (1991) Biochimie 73, 411-422. difference is that in the case of phage A repressor the axis of 4. Schnarr, M., Oertel-Buchheit, P., Kazmaier, M. & Granger- the recognition helix is almost parallel to the plane ofthe base Schnarr, M. (1991) Biochimie 73, 423-431. pairs, whereas the LexA recognition helix is expected to form 5. Little, J. W. & Hill, S. A. (1985) Proc. Natl. Acad. Sci. USA a pronounced angle with respect to the plane of the base 82, 2301-2305. 6. Hurstel, S., Granger-Schnarr, M., Daune, M. & Schnarr, M. pairs. (1986) EMBO J. 5, 793-798. A second difference resides in the fact that the LexA 7. Schnarr, M., Pouyet, J., Granger-Schnarr, M. & Daune, M. recognition helix is oriented within the major groove of the (1985) Biochemistry 24, 2812-2818. DNA such that its NH2 terminus is directed towards the 8. Schnarr, M., Granger-Schnarr, M., Hurstel, S. & Pouyet, J. palindromic center of the operator. All HTH proteins for (1988) FEBS Lett. 234, 56-60. which an x-ray structure of the complex is available are 9. Kim, B. & Little, J. W. (1992) Science 255, 203-206. 10. Dodd, I. B. & Egan, J. B. (1987) J. Mol. Biol. 194, 557-564. oriented in the opposite sense, with their NH2 termini farther 11. Lamerichs, R. M. J. N., Padilla, A., Boelens, R., Kaptein, R., away from the center than their COOH termini (30). NMR, Ottleben, G., Rfiterjans, H., Granger-Schnarr, M., Oertel, P. & genetic, and affinity cleavage data suggest, however, that lac Schnarr, M. (1989) Proc. Natl. Acad. Sci. USA 86,6863-6867. repressor (20, 31, 32) and possibly also tet repressor (33) 12. Ottleben, G., Messori, L., RUterjans, H., Kaptein, R., Granger- might be oriented with their NH2 termini directed towards the Schnarr, M. & Schnarr, M. (1991) J. Biomol. Struct. Dyn. 9, palindromic center. 447-461. 13. Wertman, K. F. & Mount, D. W. (1985) J. Bacteriol. 163, Beyond the overall positioning of the LexA recognition 376-384. helix within the major groove of the DNA, the model of the 14. Lloubes, R., Granger-Schnarr, M., Lazdunski, C. & Schnarr, LexA-DNA complex shown in Fig. 5 suggests the following M. (1991) J. Biol. Chem. 266, 2303-2312. predictions concerning the recognition of the CTGT motif: 15. Oertel-Buchheit, P., Lamerichs, R. M. J. N., Schnarr, M. & (i) Asn-41 should be located close to the adenine base ofthe Granger-Schnarr, M. (1990) Mol. Gen. Genet. 223, 40-48. second base pair ofthe CTGT motif(base pair 7), potentially 16. Sluka, J. P., Horvath, S. J., Bruist, M. F., Simon, M. I. & allowing the formation of two hydrogen bonds between the Dervan, P. B. (1987) Science 238, 1129-1132. amide group of Asn-41 and, respectively, the exocyclic NH2 17. Mack, D. P., Sluka, J. P., Shin, J. A., Griffin, J. H., Simon, M. I. & Dervan, P. B. (1990) Biochemistry 29, 6561-6567. group and the N-7 atom ofadenine 7. The methyl group ofthe 18. Oakley, M. G. & Dervan, P. B. (1990) Science 248, 847-850. opposite thymine base is also expected to play a role in LexA 19. Graham, K. S. & Dervan, P. B. (1990) J. Biol. Chem. 265, recognition, since its removal weakens DNA binding (14). 16534-16540. According to our model the formation of a hydrophobic 20. Shin, J. A., Ebright, R. H. & Dervan, P. B. (1991) Nuckic contact between this methyl group and the methyl side chain Acids Res. 19, 5233-5236. of Ala-42 would be possible upon a minor distortion of the 21. Ebright, R. H., Ebright, Y. W., Pendergast, P. S. & Gunasek- DNA structure. era, A. (1990) Proc. Natl. Acad. Sci. USA 87, 2882-2886. 22. Praseuth, D., Chassignol, M., Takasugi, M., Le Doan, T., (ii) The contacts with the third and fourth base pair of the Thuong, N. T. & H6lene, C. (1987) J. Mol. Biol. 196, 939-942. CTGT motif (base pairs 6 and 5) are more hazardous to 23. Praseuth, D., Perrouault, L., Le Doan, T., Chassignol, M., predict because these base pairs are farther from the Thuong, N. T. & Helene, C. (1988) Proc. Natl. Acad. Sci. USA crosslinking and genetic docking points. Ser-39 might contact 85, 1349-1353. the guanine base of the third base pair of the CTGT motif 24. Pendergrast, P. S., Chen, Y., Ebright, Y. W. & Ebright, R. H. upon formation of a hydrogen bond between its hydroxyl (1992) Proc. Natl. Acad. Sci. USA 89, 10287-10291. group and the N-7 atom of guanine 6. 25. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, Independently of the precise details of recognition, the 499-560. the LexA is 26. Hurstel, S., Granger-Schnarr, M. & Schnarr, M. (1988) EMBO overall positioning of recognition helix well J. 7, 269-275. established by the crosslinking and genetic data, and we may 27. Brent, R. & Ptashne, M. (1981) Proc. Natl. Acad. Sci. USA 78, conclude that the binding mode ofthe LexA recognition helix 4204-4208. is apparently different from the way the recognition helices of 28. Jordan, S. R. & Pabo, C. 0. (1988) Science 242, 893-899. classical HTH proteins are inserted into the major groove. 29. Thliveris, A. T. & Mount, D. W. (1992) Proc. Natl. Acad. Sci. USA 89, 4500-4504. We are grateful to Dr. R. H. Ebright for communicating many 30. Harison, S. C. & Aggarwal, A. A. (1990)Annu. Rev. Biochem. details concerning the cysteine-specific incorporation and the pho- 59, 933-969. tocrosslinking of phenyl azide in the case of the catabolite gene 31. Boelens, R., Scheek, R. M., van Boom, J. H. & Kaptein, R. activator protein prior to publication, to Dr. J. Reinbolt for deter- (1987) J. Mol. Biol. 193, 213-216. mining the carboxymethylcysteine content of the different LexA 32. Lehming, N., Sartorius, J., Kisters-Woike, B., von Wilcken- variants, and to Dr. R. Fogh for his introduction into the use of Bergmann, B. & MUller-Hill, B. (1990) EMBO J. 9, 615-621. INSIGHT ii. This work was supported by grants from the European 33. Wissmann, A., Baumeister, R., MUller, G., Hecht, B., Helbl, Community (ST2J-0291), the Institut National de la Sante et de la V., Pfleiderer, K. & Hillen, W. (1991) EMBO J. 10, 4145-4152. Downloaded by guest on September 25, 2021