Eur. J. Biochem. 9Y, 105-111 (1979)
Isolation of Plasmid-Protein Complexes from Escherichia coli Stephen BUSBY, Annie KOLB, and Henri BUC Departement de Biologie MolCculaire, lnstitut Pasteur, Paris (Received March 12, 1979)
A procedure is described for the isolation of complexes between pMB9 plasmids and protein from Escherichia coli which are stable during centrifugation on sucrose gradients and are not destroyed in the presence of competitor DNA. The proteins in these complexes have been analysed by dodecyl sulphate/polyacrylamide gel electrophoresis. Only 10 polypeptide species are found in significant quantities, many of which are bound to both the plasmid and host DNA. We have also detected the presence of one protein which binds to a specific DNA sequence inserted in the plasmid.
We are still far from understanding the molecular mechanism by which DNA is packaged in Escherichia coli [l 1. An ordered structure involving histone-like proteins has been proposed by several workers [2,3], and has been visualised by Griffiths’ electron microscopy observations [4]. From the biochemical point of view, it is still a matter of debate as to the number and nature of the proteins involved in DNA organisation, their spatial disposition and their interaction with sequence-specific proteins involved in transcription and replication. Investigation of these factors has been hampered by several difficulties. A synthetic approach based on the isolation of all the DNA binding proteins by affinity chromatography is precluded by the large number of protein species retained [5]. On the other hand, attempts to analyse directly the E. coli folded chromosome are complicated by the size of the host DNA and by the presence of membranes and ribosomes [6-91. To simplify the problem, Manoil et al. [lo] and Varshavsky et al. [2] have generated DNA fragments by mild DNAse treatment of E. coli lysates and subsequently isolated DNA-protein complexes by gel filtration. The former authors, experimenting with T4-infected E. coli, were able to show the presence of several T4-coded proteins in their complexes. Varshavsky et al., working with fragments from the E. coli chromosome, showed that the DNA fragments were complexed to two basic proteins of molecular weight 17000 and 9000, the latter of which was identified as the histone-like protein H U [3]. Using methods analogous to those described by the group of Helinski [ l l - 131, Manoil et al. [lo] and Varshavsky et al. [2], we have isolated plasmid-
protein complexes from E. coli. Some plasmids are especially suitable for this type of study as they are present in cells in multiple copies and are much smaller than the genomes of T4 or E. coli. Complexes with protein can be isolated without DNAse treatments. A further advantage of the use of plasmids is that insertions of specific DNA sequences can easily be made, thus permitting the detection of any protein with a strong affinity for the insert. We have used pMB9 plasmid (see [14]) containing a 203-base-pair insertion of lactose operator DNA at the EcoRI restriction site [15], and have checked for the presence of the lactose repressor in our preparations. MATERIALS AND METHODS Preparation and Fractionation of Lysates E. coli strain HBlOl [16] containing dimer pMB9 plasmid, either with or without the 203-base-pair lactose DNA insertion, was grown to an absorbance of 0.6 at 600 nm in 100 ml of low-sulphate medium (10 g/1 citric acid, 2 g/l NH4C1, 0.2 g/l MgC12, 5 mg/l FeC13, 0.68 g/1 KHzP04, 10 mg/l (NH4)2S04, 6 g/1 glucose and all amino acids [17] except cysteine and methionine), including 3.0 mCi carrier-free (3sS)su1phate (Amersham) and 0.25 mCi (32P)phosphate(Commissariat a 1’Energie Atomique, Saclay, France). After centrifugation and washing in 63 B1 medium [18], bacteria were resuspended in 0.4 ml 50 mM Tris, 40 mM EDTA, 25 o/, sucrose pH 7.5 and incubated on ice with 60 p1 of a 1-mg/ml solution of lysozyme (Worthington) for 10min. After a 6-min incubation with 1.5 pl of a 50-mg/ml solution of phenylmethylsulphonyl fluoride and 20 p1 of 0.5 M
Plasmid-Protein Complexes from E. c d i
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EDTA, 1 ml of 0.1 % Triton X-100 in 50 mM Tris, 40 mM EDTA pH 8 was added. The lysate was centrifuged in a Beckman SW56 rotor at 40000 rev./min for 20 min and 0.8 ml of the supernatant (clear lysate) was applied to a column (53 x 0.9 cm) of Biogel A 150m which was equilibrated and run at 4°C in 20 mM Tris, 60 mM NaCl, 1 mM EDTA, 0.5 mM dithioerythritol pH 7.4 containing 50 pg/ml phenylmethylsulphonyl fluoride. Fractions from the column were measured for phosphorus radioactivity by Cerenkov counting and for sulphur radioactivity with Biofluor scintillation mixture (NEN). Measurement of DNA and Protein Concentrations DNA concentrations were determined from the phosphorus radioactivity and the specific radioactivity of phosphorus used in the growth medium. Alternatively, the fluorescence of the intercalant 4',6-diamidino-2-phenyl indole was measured [19], using deproteinised plasmid DNA for calibration. The protein concentration in any sample was estimated from the sulphur radioactivity, and the specific radioactivity of protein in the clear lysate. This was calculated from direct measurements of sulphur radioactivity and the protein concentration, determined using the Lowry method [20], calibrated with bovine serum albumin. Analysis of DNA and Protein Plasmid ["PIDNA from complexes was analysed in 0.7 % agarose gels (run as in [21]) using purified dimer pMB9 plasmid DNA as a marker. The gels were dried and, after autoradiography, the DNA bands in the plasmid-protein complex were identified on Kodirex film, scanned on a Vernon apparatus and quantified. 35S-labelled protein from complexes was analysed in 10-25X acrylamide gradient gels run in the presence of sodium dodecyl sulphate according to Laemmli [22]. Gels were calibrated for molecular weight with E. coli HU (9000) [9], ox heart cytochrome c (12400) [23], whale myoglobin (17000) [23], pancreatic trypsinogen (24000) [23], E. coli RNA polymerase (165 000, 155 000,90 000 and 36 500) [24,25j, E. coli lac repressor (38 590) [26], bovine serum albumin (68000) [23], rabbit muscle phosphorylase b (97000) [27] and E. coli P-galactosidase (135000) [28]. Radioactivity in the gels was revealed using fluorography [29,30]. After scanning, the proportion of 35S in each band was deduced. Sucrose Gradient Centrifugation 400-pl samples of fractions containing plasmid and protein were applied to 11-ml 5-20% sucrose
gradients containing 20 mM Tris, 60 mM NaCl, 1 mM EDTA, 0.5 mM dithioerythritol pH 7.4 and 50 pg/ml phenylmethylsulphonyl fluoride. Centrifugation was performed at 40000 rev./min for 4 h in an SW41 Beckman rotor at 4°C. Gradients were subsequently fractionated into 0.4-ml aliquots which were counted for 32P and 3sS radioactivity. Miscellaneous Treatment of plasmid-protein samples with restriction endonuclease EcoRI was as follows : 400-p1 fractions were incubated with 4 pl M MgC12 and 5 pl EcoRI enzyme (Miles) for 1 h at 37°C. Protein-free pMB9 DNA was prepared according to Tanaka and Weisblum [31], and was phenol-extracted. RNA polymerase was purified as described by Burgess and Jendrisak [32]. Purified lactose repressor was a gift of Dr J.-C. Maurizot. Both proteins were radioactively labelled by reductive alkylation with [14C]formaldehyde [33]. pMB9 plasmid with and without the lactose operator DNA insertion was a gift from Dr R. Ogata. Lipid analyses were kindly performed by Dr B. Lubochinsky [34]. The presence of RNA in the plasmid-protein complex was tested using caesium sulphate isopycnic centrifugation [35]. RESULTS Isolation and Analysis of Plasm id-Pr o te in Fractions Cells containing dimer pMB9 plasmid were labelled and lysed as described in Materials and Methods: the clear lysate was applied to a column of Biogel A-1 50 m. Fig. 1 shows the profile of elution of such a column. A peak of protein (35S radioactivity) and 32P radioactivity emerges near the void volume of the column. Agarose gel electrophoresis shows that the fractions of this peak contain plasmid DNA. On the gel, shown in Fig.2a, the two lower bands correspond to the supercoiled and relaxed forms of the plasmid (60% of the plasmid is supercoiled and 40% is relaxed). A third band is observed, corresponding to DNA of molecular weight greater than 1O', which represents 20% of the total 32P in the plasmid-containing fractions. Nearly all the phosphorus radioactivity in these fractions corresponds to DNA since only trace quantities of phospholipid have been found (1.6 '%, of total 32P) and since we have been unable to detect RNA (less than 5 of total 32P). A small reproducible amount of the 3sS radioactivity (0.04 %) applied to the column (Fig. 1) emerges in the same fractions as the plasmid DNA. These plasmidprotein fractions contain 1.3 pg protein/pg DNA. The larger peaks of sulphur and phosphorus radioactivity which follow as the column is developed are due to the bulk of E. coli proteins and nucleic acid fragments.
S. Busby, A. Kolb, and H. Buc
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Fig. 1 . E/utiori projile from Biogc,l (.o/uwi. 0.8 nil of clear lysate from labelled bacteria containing pMBY dimer plasmid was applied to a Biogel column, as described in Materials and Methods. 400-pl fractions were collected from the column and were countcd for 32P radioactivity (a).40-pI samples were taken from each fraction and were counted for 35S radioactivity (0)
We have looked for sequence-specific DNA binding proteins in the plasmid-protein fraction by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (Fig. 3). Fig. 3 A (track b), shows the proteins present in such a fraction isolated from bacteria containing plasmid with the lactose operator DNA fragment insertion. More than 90 % of the proteins are contained in nine bands. On less exposed films, the least mobile of these bands is clearly seen to be a doublet which comigrates with the fl and fi' subunits of RNA polymerase (track d). Other bands are seen with the same electrophoretic mobilities as the CJ and a subunits of RNA polymerase and the purified lactose repressor (track a). Further evidence that this latter band is indeed the lactose repressor is seen in track (c) of Fig. 3A. Here, we show analysis of the proteins in the plasmid-protein fraction, which had been isolated on a column equilibrated with 1 mM isopropylthiogalactoside. The major difference with track (b) is the total lack of the band that comigrates with the purified lactose repressor. This difference is further shown in Fig. 3 B, where the autoradiogram has been deliberately overexposed. As isopropylthiogalactoside is a specific inducer of the lactose operon and is known to prevent repressor binding to operator
Fig. 2. Agrirasr gel elec~trophoretic analysis q / D N A . Analysis of [32P]DNA in the DNA-protein fractions isolated near the void volume of the Biogel column, run with lysates of (a) bacteria containing dimeric pMBY plasmid or (b) bacteria containing no plasmid. Analysis was performed in 0.7% agarose gels and ["'PID N A bands were revealed by autoradiography. Thc positions corresponding to relaxed and supcrcoilcd purified dimer pMB9 D N A are marked
DNA [36,37], we conclude that the 39000-molecularweight protein band removed by isopropylthiogalactoside is the lactose repressor. In order to check that the presence of the lactose repressor in our preparations is due to the 203-basepair lactose operator DNA insertion, we have compared the proteins in preparations of the plasmidprotein fraction from bacteria containing pMB9 plasmid either without or with the lactose operator insertion (Fig. 3 C). The only significant difference between the two preparations is the presence in the latter case of the band which comigrates with the lactose repressor. Apart from RNA polymerase and lactose repressor, the plasmid-protein fraction contains bands corresponding to molecular weights of 70000, 30000, 17000, 15000 and 9000. The 15000-molecular-weight band is the most intense (Fig.3A and Table 1). Experiments with Bacteria Containing N o Plasmid
We have radioactively contain any described in
performed control experiments using labelled HBlOl bacteria which do not plasmid, grown and fractionated as Materials and Methods for cells con-
Plasmid-Protein Complexes from E. coli
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Fig. 3. Analysis of 35S-/uhc//cdp~oteins in p/lsmic/-l,,-otcin/r-uctior2.~.Autoradiograms of gels treated by fluorography are shown. (A) (a) Purified 14C-labelled lactose repressor; (b) proteins in plasmid-protein fraction from bacteria containing pMB9 plasmid with lactose operator insertion; (c) proteins in plasmid-protein fraction from bacteria containing pMB9 plasmid with lactose operator insertion with 1 mM isopropylthiogalactoside included in the running buffer of the Biogel column; (d) purified I4C-labelled RNA polymerase. The calibration scale for molecular weight was determined as described in Materials and Methods. (B) (a) As A (b) but overexposed; (b) as A (c) but overexposed. (C) Proteins in plasmid-protein fraction from bacteria containing pMB9 plasmid either (a) with the lactose operator insertion or (b) without the lactose operator insertion. Note that the calibration scale in C is different to that in A and B Table 1. Percentage of 35Sradioactivity in proteins in DNA-protein fractions 3SS-labelled proteins in DNA-protein fractions were analysed o n gels as in Fig. 3. Autoradiograms of the gels were scanned and the percentage of the total radioactivity in each band was calculated. The proteins analysed were either (A) in plasmid-protein fraction from bacteria containing pMB9 plasmid with lactose operator insertion (1.3 pg protein/pg DNA) or (B) in DNA-protein fraction from bacteria containing no plasmid (1 .O pg protein/pg DNA). Results are the average of three (A) or two (B) experiments ~~
Protein
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taining plasmids. Fig. 4 A and B respectively compare the appearance of 32P and 35Snear the void volume of the column when bacteria with or without plasmid were used. Compared to the profile obtained from bacteria with plasmids, smaller peaks of radioactivity are seen with both isotopes, representing 25% of the
radioactivity obtained in the presence of the plasmid. Again, the 32P is mainly due to DNA. Agarose gel electrophoresis (Fig. 2 b) shows that this material corresponds to the upper non-plasmid band seen in Fig. 2 a and is due to sheared chromosomal DNA. The fractions eluting near the void volume of the column contain 1 pg protein/pg DNA. Analysis of the protein in the polyacrylamide gels shows that 70 % of the protein corresponds to bands of molecular weight 30 000, 15000 and 9000 (Table 1). Little RNA polymerase and no lactose repressor is found and, again, the 15000-molecular-weight band is the most intense.
Evidence for a Complex between D N A and Protein The plasmid-protein fraction contains some proteins which have a known affinity for the plasmid (the lactose repressor and RNA polymerase). Furthermore, from the increase in the total protein recovered in the DNA-protein fraction when cells harbouring the plasmid are used (Fig. 4), we can infer that 75 % of the protein in this fraction is present by virtue of its affinity for the plasmid. Sucrose gradient sedimentation analyses have been performed in order to show that this fraction contains a true complex between DNA and protein. In Fig.5A and B it is shown that DNA and protein cosediment both before and after treatment with EcoRI restriction endonuclease. The 10 protein
S. Busby, A. Kolb, and H. Buc
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Fig. 4. Comparison 01 column elution profile f o r lysares with or n~irhoutplasniid. 0.8 ml ol' clear lysate from labelled bacteria either containing (0, 0 ) or without (0, m) pMB9 plasmid dimer was applied to a Biogel column, as described in Materials and Methods, and as in Fig. 1 . (A) 400-p1 fractions were collected and counted for "P radioactivity (0,W). (B) 40 p1 were taken from each fraction and counted for 35S radioactivity (0, 0). Only the part of the elution profile near the excluded volume of the column is shown
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Fig. 5. Sucrose gradient centrifugutiort of DNA-protein fructions. 400-pl samples were applied to sucrose gradients. After centrifugation, 400-pl fractions were counted for 32P radioactivity (0,N). 40-pi aliquots of each fraction were counted for 35S radioactivity (0, 0).The samples applied to the gradients were as follows. (A) Plasmid-protein fraction from bacteria containing dimer pMB9 plasmid. (B) Plasmid-protein fraction from bacteria containing dimer pMB9 plasmid after EcoRI treatment. (C) Purified dimer supercoiled and relaxed pMB9 plasmid (A); EcoRI fragment of pMB9 D N A ( x ); the absorbance at 260 nm of each fraction is plotted and the positions of sedimentation of E. coli 5 0 4 and 3 0 3 ribosomal subunits and 2 3 3 rRNA are also shown for reference. (D) DNA-protein fraction from bacteria containing no plasmid. (E) DNA-protein fraction from bacteria containing no plasmid after EcoRI treatment. The direction of sedimentation is from left to right. The data shown in D or E and that in A or B are directly comparable. The specific activity of the 32Pincorporated in DNA and total quantity of chromosomal D N A is the same in the four figures. Between 65 % and 75 % of the 35Sapplied to the gradients were recovered whereas the recovery of 3zP was greater than 85
110
species seen in the fraction applied to the gradient (Fig. 3 A) were again present in the complexes isolated after sucrose gradient centrifugation (data not shown). Fig. 5 C shows the sedimentation properties of deproteinised pMB9 plasmid DNA both before and after EcoRI treatment. The proteins in the plasmid-protein fraction clearly displace the DNA in the sucrose gradients. Fig.5D and E show the sedimentation profile of the DNA-protein fraction obtained from the control experiment using cells without plasmid, both before and after EcoRI treatment. In both cases the 35Slabelled protein cosediments with the DNA. This protein, consisting mainly of the 30 000-M,, 15000-M, and 9000-M, polypeptides is common to the plasmidprotein fraction. We have compared the amounts of these polypeptides in the DNA protein fraction both in the presence and absence of plasmid (Table 1). Taking into account the fact that the chromosomal contaminant represents 20% of the DNA in the plasmid-protein fraction, we can deduce that the 30000-M,, 15000-M, and 9000-M, polypeptides are bound both to the plasmid and to the chromosome. A similar argument applies for RNA polymerase. We have attempted to challenge the protein in the complexes shown in Fig.5 with an excess of uncomplexed DNA. In the presence of a 40-fold weight excess of non-radioactive monomer plasmid DNA, only 15% of the protein is displaced from the dimer plasmid onto the monomer (data not shown). This demonstrates that the complex we have isolated has an intrinsic kinetic stability. The time of preincubation with competitor DNA (up to 20 min at 37"C), the nature of the competitor DNA (supercoiled, relaxed or linear) and the salt concentration in the gradient (up to 200mM NaCl) do not affect this result.
DISCUSSION Mild methods have been used to isolate complexes of protein and plasmid from E. coli lysates. These preparations differ from previously described proteinD N A complexes. The group of Helinski [l 1 - 131 has analysed complexes of plasmids with small amounts of protein. In contrast, we describe here complexes containing roughly equivalent weights of protein and DNA. Unlike folded chromosomes [6-91, our preparations contain only trace quantities of membrane and RNA and the protein consists of relatively few species. Our experimental conditions are most directly comparable with those of Varshavsky et al. [2] who found 0.2 pg protein complexed/pg fragmented chromosomal DNA. Whereas these authors observed mainly proteins of molecular weight 17000 and 9000,
Plasmid-Protein Complexes from E. coli
we find a more complex protein pattern consisting of small polypeptides of molecular weight 30000, 17000, 15000 and 9000.We do not know, as yet, if any of these species are identical to the proteins seen by Varshavsky et al. [2]. The most abundant polypeptide is the 15000-M, species, which represents 25% of the total 35Sin the complexes (Table 1). In more-resolving sodium dodecyl sulphate/urea gels, the 15000-M, protein comigrates with HI, a thermostable DNA binding protein, which is found in large amounts in E. coli [38,39]. The differences between our results and those of Varshavsky et al. [2] may be due either to different labelling conditions or to the fact that we have not used DNAse treatments. Analysis of our preparations is complicated by two problems. Firstly, because of the small amount of material which can be isolated, we have been forced to use 35Slabelling of proteins. Variations from the average in the sulphur content of proteins in the clear lysate will distort the weight ratio of protein to DNA calculated for the plasmid-protein fraction. For example, as RNA polymerase, a major component of the complexes, is relatively poor in sulphur [24], the weight ratio of protein to DNA in fractions is likely to be underestimated. Additionally, the relative quantities of the different protein species may be distorted and proteins which contain no sulphur may remain undetected. The second complication is that we are unable to completely eliminate the 20 % contamination of sheared chromosomal DNA from the pkdsmidprotein fraction, even after sucrose gradient centrifugation. We find, however, that the proteins complexed to this DNA are indistinguishable from those bound to the plasmid. Despite these complications, we have unambiguously identified the lactose repressor in the plasmidprotein fractions when the plasmid contains insertions of lactose operator DNA. In this particular case, we know the sulphur content of the protein (40) and we can calculate that 1.4- 2.0 repressor monomers are present per 203-base-pair lac DNA fragment. As the repressor acts as a tetramer [41], we deduce that, at most, 50% of the lactose insertions bind the repressor. X-gal indicator plates [18j confirm that, in bacteria containing plasmid with the insertions, insufficient repressor is present to saturate all the lactose operators and the P-galactosidase gene in the host DNA is derepressed. In principle, the method which we have described can allow rapid identification of regulatory proteins at other cloned operator sequences. The limitation of this is, however, that many such proteins may bind too weakly to be retained in the plasmid-protein fraction of the Biogel column. What is the significance and function of the other proteins which are consistently found attached to the plasmid and the fragments of chromosomal DNA? The presence of RNA polymerase (and the lactose
S . Busby, A. Kolb, and H . Buc
repressor in the appropriate cases), as well as the stability of the complexes when challenged with other DNA, argues that these proteins bind to the plasmid in vivo. Varshavsky et al. [2] suggest that the proteins associated with fragments of DNA may be responsible for the nucleosome-like structures seen on bacterial DNA [4]. Some of the proteins which we find associated with DNA may play a similar role and are present in a quantity sufficient to fulfill such a function. Further study of the plasniid-protein cornplex should give information about the mechanisms by which bacterial DNA is condensed; it is also likely that this complex will be suitable material for an investigation of the -factors dictating transcriptional specificity. We acknowledge the Centre National de la Recherche Scientifiyur (L.A. 270), the DPlPgation GPnPrale a la Recherche Scientifique et Technique (grant M R M 76.7.1 191) and the hstitut National de la Sunti et de la Recherche Midicale (grant 77.84) for financial support. We thank Drs A. Spassky, J. Yaniv and N . Kjeldgaard for helpful discussions, and Drs A.-L. Haenni and A. Ullmann for constructive criticisms of the manuscript.
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111 14. Meagher, R. B., Tait, R. C., Betlach, M. & Boyer, H. W . (1977) Cell 10, 521 -536. 15. Ogata, R. &Gilbert, W. (1977) Proc. NatlAcad. Sci. U.S.A. 74, 4973 -4976. 16. Boyer, H . W. & Roulland-Dussoix, D. (1969) J . Mol. Biol. 41, 459 - 472. 17. Novick, R. P. & Maas, W. K. (1961) J . Bacteriol. 81, 236-240. 18. Miller, J. H . (1972) Experiments in Molecular Genrtic,s, Cold Spring Harbour Laboratory, New York. 19. KapuScinski, J. & Skoczylas, B. (1977) Anal. Biochem. 83, 252 - 257. 20. Lowry, 0. H., Rosebrough, N . J., Farr, A. L. & Randall, R. J. (1951) J . Biol. Chem. 193, 265-275. 21. Meyers, J. A., Sanchez, D., Elwell, L. P. & Falkow, S. (1976) J . Bacteriol. 127, 1529- 1533. 22. Laemmli, U. K. (1970) Nature (Lond.) 227, 680-685. 23. Sober, H. A. (ed.) (1968) Handbook qf Biochemistry: Selected Data ,for Molecular Biology, section C, pp. C1 -C193, The Chemical Rubber Co, Cleveland. 24. Burgess, R. R. (1976) in R N A Polymerase (Losick, R. C? Chamberlin, M., eds) pp. 69- 100, Cold Spring Harbour Laboratory, New York. 25. Ovchinnikov, Y. A,, Lipkin, V. M., Modyanov, N . N., Chertov, 0. Y . & Smirnov, Y. V. (1977) FEBS Lett. 76, 108- 11 1. 26. Beyreuther, K. (1978) Nature (Lond.) 274, 767. 27. Titani, K., Koide, A,, Hermann, J., Ericsson, L. H., Kumar, S., Wade, R. D., Walsh, K. A., Neurath, H. & Fischer, E. H. (1977) Proc. Nut1 Acad. Sci. U.S.A. 74, 4762-4766. 28. Zabin, I. & Fowler, A. V. (1970) in The Lactose Operon (Beckwith, J. R. & Zipser, D., eds) pp. 27-47, Cold Spring Harbour Laboratory, New York. 29. Bonner, W. M . & Laskey, R. A. (1974) Eur. J . Biochem. 46, 83-88. 30. Laskey, R. A. & Mills, A. D. (1975) Eur. J . Biochem. 56, 335 - 341. 31. Tanaka, T. & Weisblum, B. (1975) J . Bacteriol. 121, 354-362. 32. Burgess, R. R. & Jendrisak, J. J. (1975) Biochemistry, 14,46344638. 33. Means, G. E. & Feeney, R . E. (1971) in Clzemical Modr/i'cation of Proteins, p. 216, Holden-Day, San Francisco. 34. Rigomier, D. & Lubochinsky, B. (1974) Ann. Microhiol. (Paris) 12SB, 295 - 303. 35 Zolotor, L. & Engler, R. (1967) Biochim. Biophys. Acta, 145, 52 - 59. 36. Gilbert, W., Gralla, J., Majors, J. Sr Maxam, A. (1975) in Protein Ligand Interactions (Sund, H . & Blauer, G., eds) pp. 193-210, de Gruyter, Berlin. 37. Von Hippel, P. H., Revzin, A,, Gross, C. A. & Wang, A. C. (1975) in Protein Ligand Interactions (Sund, H. & Blauer, G., eds) pp. 270-288, de Gruyter, Berlin. 38. Jacquet, M., Cukier-Kahn, R., Pla, J. & Gros, F. (1971) Biochem. Biophys. Res. Commun. 45, 1597- 1607. 39 Spassky, A. & Buc, H. C. (1977) Eur. J . Biochem. 81, 79-90. 40 Farabaugh, P. J. (1978) Nature (Lond.) 274, 765-769. 41, Bourgeois, S. & Pfahl, M. (1976) Adv. Protein Cliem. 30, 1-99.
S. Busby, A. Kolb, and H. Buc*, Departement de Biologie Moleculaire, Institut Pasteur, 25/28 Rue du Docteur-Roux, F-75724 Paris-Cedex-15, France
* To whom correspondence should be addressed.
Isolation of Plasmid-Protein Complexes from Escherichia coli Stephen BUSBY, Annie KOLB, and Henri BUC Departement de Biologie MolCculaire, lnstitut Pasteur, Paris (Received March 12, 1979)
A procedure is described for the isolation of complexes between pMB9 plasmids and protein from Escherichia coli which are stable during centrifugation on sucrose gradients and are not destroyed in the presence of competitor DNA. The proteins in these complexes have been analysed by dodecyl sulphate/polyacrylamide gel electrophoresis. Only 10 polypeptide species are found in significant quantities, many of which are bound to both the plasmid and host DNA. We have also detected the presence of one protein which binds to a specific DNA sequence inserted in the plasmid.
We are still far from understanding the molecular mechanism by which DNA is packaged in Escherichia coli [l 1. An ordered structure involving histone-like proteins has been proposed by several workers [2,3], and has been visualised by Griffiths’ electron microscopy observations [4]. From the biochemical point of view, it is still a matter of debate as to the number and nature of the proteins involved in DNA organisation, their spatial disposition and their interaction with sequence-specific proteins involved in transcription and replication. Investigation of these factors has been hampered by several difficulties. A synthetic approach based on the isolation of all the DNA binding proteins by affinity chromatography is precluded by the large number of protein species retained [5]. On the other hand, attempts to analyse directly the E. coli folded chromosome are complicated by the size of the host DNA and by the presence of membranes and ribosomes [6-91. To simplify the problem, Manoil et al. [lo] and Varshavsky et al. [2] have generated DNA fragments by mild DNAse treatment of E. coli lysates and subsequently isolated DNA-protein complexes by gel filtration. The former authors, experimenting with T4-infected E. coli, were able to show the presence of several T4-coded proteins in their complexes. Varshavsky et al., working with fragments from the E. coli chromosome, showed that the DNA fragments were complexed to two basic proteins of molecular weight 17000 and 9000, the latter of which was identified as the histone-like protein H U [3]. Using methods analogous to those described by the group of Helinski [ l l - 131, Manoil et al. [lo] and Varshavsky et al. [2], we have isolated plasmid-
protein complexes from E. coli. Some plasmids are especially suitable for this type of study as they are present in cells in multiple copies and are much smaller than the genomes of T4 or E. coli. Complexes with protein can be isolated without DNAse treatments. A further advantage of the use of plasmids is that insertions of specific DNA sequences can easily be made, thus permitting the detection of any protein with a strong affinity for the insert. We have used pMB9 plasmid (see [14]) containing a 203-base-pair insertion of lactose operator DNA at the EcoRI restriction site [15], and have checked for the presence of the lactose repressor in our preparations. MATERIALS AND METHODS Preparation and Fractionation of Lysates E. coli strain HBlOl [16] containing dimer pMB9 plasmid, either with or without the 203-base-pair lactose DNA insertion, was grown to an absorbance of 0.6 at 600 nm in 100 ml of low-sulphate medium (10 g/1 citric acid, 2 g/l NH4C1, 0.2 g/l MgC12, 5 mg/l FeC13, 0.68 g/1 KHzP04, 10 mg/l (NH4)2S04, 6 g/1 glucose and all amino acids [17] except cysteine and methionine), including 3.0 mCi carrier-free (3sS)su1phate (Amersham) and 0.25 mCi (32P)phosphate(Commissariat a 1’Energie Atomique, Saclay, France). After centrifugation and washing in 63 B1 medium [18], bacteria were resuspended in 0.4 ml 50 mM Tris, 40 mM EDTA, 25 o/, sucrose pH 7.5 and incubated on ice with 60 p1 of a 1-mg/ml solution of lysozyme (Worthington) for 10min. After a 6-min incubation with 1.5 pl of a 50-mg/ml solution of phenylmethylsulphonyl fluoride and 20 p1 of 0.5 M
Plasmid-Protein Complexes from E. c d i
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EDTA, 1 ml of 0.1 % Triton X-100 in 50 mM Tris, 40 mM EDTA pH 8 was added. The lysate was centrifuged in a Beckman SW56 rotor at 40000 rev./min for 20 min and 0.8 ml of the supernatant (clear lysate) was applied to a column (53 x 0.9 cm) of Biogel A 150m which was equilibrated and run at 4°C in 20 mM Tris, 60 mM NaCl, 1 mM EDTA, 0.5 mM dithioerythritol pH 7.4 containing 50 pg/ml phenylmethylsulphonyl fluoride. Fractions from the column were measured for phosphorus radioactivity by Cerenkov counting and for sulphur radioactivity with Biofluor scintillation mixture (NEN). Measurement of DNA and Protein Concentrations DNA concentrations were determined from the phosphorus radioactivity and the specific radioactivity of phosphorus used in the growth medium. Alternatively, the fluorescence of the intercalant 4',6-diamidino-2-phenyl indole was measured [19], using deproteinised plasmid DNA for calibration. The protein concentration in any sample was estimated from the sulphur radioactivity, and the specific radioactivity of protein in the clear lysate. This was calculated from direct measurements of sulphur radioactivity and the protein concentration, determined using the Lowry method [20], calibrated with bovine serum albumin. Analysis of DNA and Protein Plasmid ["PIDNA from complexes was analysed in 0.7 % agarose gels (run as in [21]) using purified dimer pMB9 plasmid DNA as a marker. The gels were dried and, after autoradiography, the DNA bands in the plasmid-protein complex were identified on Kodirex film, scanned on a Vernon apparatus and quantified. 35S-labelled protein from complexes was analysed in 10-25X acrylamide gradient gels run in the presence of sodium dodecyl sulphate according to Laemmli [22]. Gels were calibrated for molecular weight with E. coli HU (9000) [9], ox heart cytochrome c (12400) [23], whale myoglobin (17000) [23], pancreatic trypsinogen (24000) [23], E. coli RNA polymerase (165 000, 155 000,90 000 and 36 500) [24,25j, E. coli lac repressor (38 590) [26], bovine serum albumin (68000) [23], rabbit muscle phosphorylase b (97000) [27] and E. coli P-galactosidase (135000) [28]. Radioactivity in the gels was revealed using fluorography [29,30]. After scanning, the proportion of 35S in each band was deduced. Sucrose Gradient Centrifugation 400-pl samples of fractions containing plasmid and protein were applied to 11-ml 5-20% sucrose
gradients containing 20 mM Tris, 60 mM NaCl, 1 mM EDTA, 0.5 mM dithioerythritol pH 7.4 and 50 pg/ml phenylmethylsulphonyl fluoride. Centrifugation was performed at 40000 rev./min for 4 h in an SW41 Beckman rotor at 4°C. Gradients were subsequently fractionated into 0.4-ml aliquots which were counted for 32P and 3sS radioactivity. Miscellaneous Treatment of plasmid-protein samples with restriction endonuclease EcoRI was as follows : 400-p1 fractions were incubated with 4 pl M MgC12 and 5 pl EcoRI enzyme (Miles) for 1 h at 37°C. Protein-free pMB9 DNA was prepared according to Tanaka and Weisblum [31], and was phenol-extracted. RNA polymerase was purified as described by Burgess and Jendrisak [32]. Purified lactose repressor was a gift of Dr J.-C. Maurizot. Both proteins were radioactively labelled by reductive alkylation with [14C]formaldehyde [33]. pMB9 plasmid with and without the lactose operator DNA insertion was a gift from Dr R. Ogata. Lipid analyses were kindly performed by Dr B. Lubochinsky [34]. The presence of RNA in the plasmid-protein complex was tested using caesium sulphate isopycnic centrifugation [35]. RESULTS Isolation and Analysis of Plasm id-Pr o te in Fractions Cells containing dimer pMB9 plasmid were labelled and lysed as described in Materials and Methods: the clear lysate was applied to a column of Biogel A-1 50 m. Fig. 1 shows the profile of elution of such a column. A peak of protein (35S radioactivity) and 32P radioactivity emerges near the void volume of the column. Agarose gel electrophoresis shows that the fractions of this peak contain plasmid DNA. On the gel, shown in Fig.2a, the two lower bands correspond to the supercoiled and relaxed forms of the plasmid (60% of the plasmid is supercoiled and 40% is relaxed). A third band is observed, corresponding to DNA of molecular weight greater than 1O', which represents 20% of the total 32P in the plasmid-containing fractions. Nearly all the phosphorus radioactivity in these fractions corresponds to DNA since only trace quantities of phospholipid have been found (1.6 '%, of total 32P) and since we have been unable to detect RNA (less than 5 of total 32P). A small reproducible amount of the 3sS radioactivity (0.04 %) applied to the column (Fig. 1) emerges in the same fractions as the plasmid DNA. These plasmidprotein fractions contain 1.3 pg protein/pg DNA. The larger peaks of sulphur and phosphorus radioactivity which follow as the column is developed are due to the bulk of E. coli proteins and nucleic acid fragments.
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Fig. 1 . E/utiori projile from Biogc,l (.o/uwi. 0.8 nil of clear lysate from labelled bacteria containing pMBY dimer plasmid was applied to a Biogel column, as described in Materials and Methods. 400-pl fractions were collected from the column and were countcd for 32P radioactivity (a).40-pI samples were taken from each fraction and were counted for 35S radioactivity (0)
We have looked for sequence-specific DNA binding proteins in the plasmid-protein fraction by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (Fig. 3). Fig. 3 A (track b), shows the proteins present in such a fraction isolated from bacteria containing plasmid with the lactose operator DNA fragment insertion. More than 90 % of the proteins are contained in nine bands. On less exposed films, the least mobile of these bands is clearly seen to be a doublet which comigrates with the fl and fi' subunits of RNA polymerase (track d). Other bands are seen with the same electrophoretic mobilities as the CJ and a subunits of RNA polymerase and the purified lactose repressor (track a). Further evidence that this latter band is indeed the lactose repressor is seen in track (c) of Fig. 3A. Here, we show analysis of the proteins in the plasmid-protein fraction, which had been isolated on a column equilibrated with 1 mM isopropylthiogalactoside. The major difference with track (b) is the total lack of the band that comigrates with the purified lactose repressor. This difference is further shown in Fig. 3 B, where the autoradiogram has been deliberately overexposed. As isopropylthiogalactoside is a specific inducer of the lactose operon and is known to prevent repressor binding to operator
Fig. 2. Agrirasr gel elec~trophoretic analysis q / D N A . Analysis of [32P]DNA in the DNA-protein fractions isolated near the void volume of the Biogel column, run with lysates of (a) bacteria containing dimeric pMBY plasmid or (b) bacteria containing no plasmid. Analysis was performed in 0.7% agarose gels and ["'PID N A bands were revealed by autoradiography. Thc positions corresponding to relaxed and supcrcoilcd purified dimer pMB9 D N A are marked
DNA [36,37], we conclude that the 39000-molecularweight protein band removed by isopropylthiogalactoside is the lactose repressor. In order to check that the presence of the lactose repressor in our preparations is due to the 203-basepair lactose operator DNA insertion, we have compared the proteins in preparations of the plasmidprotein fraction from bacteria containing pMB9 plasmid either without or with the lactose operator insertion (Fig. 3 C). The only significant difference between the two preparations is the presence in the latter case of the band which comigrates with the lactose repressor. Apart from RNA polymerase and lactose repressor, the plasmid-protein fraction contains bands corresponding to molecular weights of 70000, 30000, 17000, 15000 and 9000. The 15000-molecular-weight band is the most intense (Fig.3A and Table 1). Experiments with Bacteria Containing N o Plasmid
We have radioactively contain any described in
performed control experiments using labelled HBlOl bacteria which do not plasmid, grown and fractionated as Materials and Methods for cells con-
Plasmid-Protein Complexes from E. coli
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Fig. 3. Analysis of 35S-/uhc//cdp~oteins in p/lsmic/-l,,-otcin/r-uctior2.~.Autoradiograms of gels treated by fluorography are shown. (A) (a) Purified 14C-labelled lactose repressor; (b) proteins in plasmid-protein fraction from bacteria containing pMB9 plasmid with lactose operator insertion; (c) proteins in plasmid-protein fraction from bacteria containing pMB9 plasmid with lactose operator insertion with 1 mM isopropylthiogalactoside included in the running buffer of the Biogel column; (d) purified I4C-labelled RNA polymerase. The calibration scale for molecular weight was determined as described in Materials and Methods. (B) (a) As A (b) but overexposed; (b) as A (c) but overexposed. (C) Proteins in plasmid-protein fraction from bacteria containing pMB9 plasmid either (a) with the lactose operator insertion or (b) without the lactose operator insertion. Note that the calibration scale in C is different to that in A and B Table 1. Percentage of 35Sradioactivity in proteins in DNA-protein fractions 3SS-labelled proteins in DNA-protein fractions were analysed o n gels as in Fig. 3. Autoradiograms of the gels were scanned and the percentage of the total radioactivity in each band was calculated. The proteins analysed were either (A) in plasmid-protein fraction from bacteria containing pMB9 plasmid with lactose operator insertion (1.3 pg protein/pg DNA) or (B) in DNA-protein fraction from bacteria containing no plasmid (1 .O pg protein/pg DNA). Results are the average of three (A) or two (B) experiments ~~
Protein
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taining plasmids. Fig. 4 A and B respectively compare the appearance of 32P and 35Snear the void volume of the column when bacteria with or without plasmid were used. Compared to the profile obtained from bacteria with plasmids, smaller peaks of radioactivity are seen with both isotopes, representing 25% of the
radioactivity obtained in the presence of the plasmid. Again, the 32P is mainly due to DNA. Agarose gel electrophoresis (Fig. 2 b) shows that this material corresponds to the upper non-plasmid band seen in Fig. 2 a and is due to sheared chromosomal DNA. The fractions eluting near the void volume of the column contain 1 pg protein/pg DNA. Analysis of the protein in the polyacrylamide gels shows that 70 % of the protein corresponds to bands of molecular weight 30 000, 15000 and 9000 (Table 1). Little RNA polymerase and no lactose repressor is found and, again, the 15000-molecular-weight band is the most intense.
Evidence for a Complex between D N A and Protein The plasmid-protein fraction contains some proteins which have a known affinity for the plasmid (the lactose repressor and RNA polymerase). Furthermore, from the increase in the total protein recovered in the DNA-protein fraction when cells harbouring the plasmid are used (Fig. 4), we can infer that 75 % of the protein in this fraction is present by virtue of its affinity for the plasmid. Sucrose gradient sedimentation analyses have been performed in order to show that this fraction contains a true complex between DNA and protein. In Fig.5A and B it is shown that DNA and protein cosediment both before and after treatment with EcoRI restriction endonuclease. The 10 protein
S. Busby, A. Kolb, and H. Buc
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Fig. 5. Sucrose gradient centrifugutiort of DNA-protein fructions. 400-pl samples were applied to sucrose gradients. After centrifugation, 400-pl fractions were counted for 32P radioactivity (0,N). 40-pi aliquots of each fraction were counted for 35S radioactivity (0, 0).The samples applied to the gradients were as follows. (A) Plasmid-protein fraction from bacteria containing dimer pMB9 plasmid. (B) Plasmid-protein fraction from bacteria containing dimer pMB9 plasmid after EcoRI treatment. (C) Purified dimer supercoiled and relaxed pMB9 plasmid (A); EcoRI fragment of pMB9 D N A ( x ); the absorbance at 260 nm of each fraction is plotted and the positions of sedimentation of E. coli 5 0 4 and 3 0 3 ribosomal subunits and 2 3 3 rRNA are also shown for reference. (D) DNA-protein fraction from bacteria containing no plasmid. (E) DNA-protein fraction from bacteria containing no plasmid after EcoRI treatment. The direction of sedimentation is from left to right. The data shown in D or E and that in A or B are directly comparable. The specific activity of the 32Pincorporated in DNA and total quantity of chromosomal D N A is the same in the four figures. Between 65 % and 75 % of the 35Sapplied to the gradients were recovered whereas the recovery of 3zP was greater than 85
110
species seen in the fraction applied to the gradient (Fig. 3 A) were again present in the complexes isolated after sucrose gradient centrifugation (data not shown). Fig. 5 C shows the sedimentation properties of deproteinised pMB9 plasmid DNA both before and after EcoRI treatment. The proteins in the plasmid-protein fraction clearly displace the DNA in the sucrose gradients. Fig.5D and E show the sedimentation profile of the DNA-protein fraction obtained from the control experiment using cells without plasmid, both before and after EcoRI treatment. In both cases the 35Slabelled protein cosediments with the DNA. This protein, consisting mainly of the 30 000-M,, 15000-M, and 9000-M, polypeptides is common to the plasmidprotein fraction. We have compared the amounts of these polypeptides in the DNA protein fraction both in the presence and absence of plasmid (Table 1). Taking into account the fact that the chromosomal contaminant represents 20% of the DNA in the plasmid-protein fraction, we can deduce that the 30000-M,, 15000-M, and 9000-M, polypeptides are bound both to the plasmid and to the chromosome. A similar argument applies for RNA polymerase. We have attempted to challenge the protein in the complexes shown in Fig.5 with an excess of uncomplexed DNA. In the presence of a 40-fold weight excess of non-radioactive monomer plasmid DNA, only 15% of the protein is displaced from the dimer plasmid onto the monomer (data not shown). This demonstrates that the complex we have isolated has an intrinsic kinetic stability. The time of preincubation with competitor DNA (up to 20 min at 37"C), the nature of the competitor DNA (supercoiled, relaxed or linear) and the salt concentration in the gradient (up to 200mM NaCl) do not affect this result.
DISCUSSION Mild methods have been used to isolate complexes of protein and plasmid from E. coli lysates. These preparations differ from previously described proteinD N A complexes. The group of Helinski [l 1 - 131 has analysed complexes of plasmids with small amounts of protein. In contrast, we describe here complexes containing roughly equivalent weights of protein and DNA. Unlike folded chromosomes [6-91, our preparations contain only trace quantities of membrane and RNA and the protein consists of relatively few species. Our experimental conditions are most directly comparable with those of Varshavsky et al. [2] who found 0.2 pg protein complexed/pg fragmented chromosomal DNA. Whereas these authors observed mainly proteins of molecular weight 17000 and 9000,
Plasmid-Protein Complexes from E. coli
we find a more complex protein pattern consisting of small polypeptides of molecular weight 30000, 17000, 15000 and 9000.We do not know, as yet, if any of these species are identical to the proteins seen by Varshavsky et al. [2]. The most abundant polypeptide is the 15000-M, species, which represents 25% of the total 35Sin the complexes (Table 1). In more-resolving sodium dodecyl sulphate/urea gels, the 15000-M, protein comigrates with HI, a thermostable DNA binding protein, which is found in large amounts in E. coli [38,39]. The differences between our results and those of Varshavsky et al. [2] may be due either to different labelling conditions or to the fact that we have not used DNAse treatments. Analysis of our preparations is complicated by two problems. Firstly, because of the small amount of material which can be isolated, we have been forced to use 35Slabelling of proteins. Variations from the average in the sulphur content of proteins in the clear lysate will distort the weight ratio of protein to DNA calculated for the plasmid-protein fraction. For example, as RNA polymerase, a major component of the complexes, is relatively poor in sulphur [24], the weight ratio of protein to DNA in fractions is likely to be underestimated. Additionally, the relative quantities of the different protein species may be distorted and proteins which contain no sulphur may remain undetected. The second complication is that we are unable to completely eliminate the 20 % contamination of sheared chromosomal DNA from the pkdsmidprotein fraction, even after sucrose gradient centrifugation. We find, however, that the proteins complexed to this DNA are indistinguishable from those bound to the plasmid. Despite these complications, we have unambiguously identified the lactose repressor in the plasmidprotein fractions when the plasmid contains insertions of lactose operator DNA. In this particular case, we know the sulphur content of the protein (40) and we can calculate that 1.4- 2.0 repressor monomers are present per 203-base-pair lac DNA fragment. As the repressor acts as a tetramer [41], we deduce that, at most, 50% of the lactose insertions bind the repressor. X-gal indicator plates [18j confirm that, in bacteria containing plasmid with the insertions, insufficient repressor is present to saturate all the lactose operators and the P-galactosidase gene in the host DNA is derepressed. In principle, the method which we have described can allow rapid identification of regulatory proteins at other cloned operator sequences. The limitation of this is, however, that many such proteins may bind too weakly to be retained in the plasmid-protein fraction of the Biogel column. What is the significance and function of the other proteins which are consistently found attached to the plasmid and the fragments of chromosomal DNA? The presence of RNA polymerase (and the lactose
S . Busby, A. Kolb, and H . Buc
repressor in the appropriate cases), as well as the stability of the complexes when challenged with other DNA, argues that these proteins bind to the plasmid in vivo. Varshavsky et al. [2] suggest that the proteins associated with fragments of DNA may be responsible for the nucleosome-like structures seen on bacterial DNA [4]. Some of the proteins which we find associated with DNA may play a similar role and are present in a quantity sufficient to fulfill such a function. Further study of the plasniid-protein cornplex should give information about the mechanisms by which bacterial DNA is condensed; it is also likely that this complex will be suitable material for an investigation of the -factors dictating transcriptional specificity. We acknowledge the Centre National de la Recherche Scientifiyur (L.A. 270), the DPlPgation GPnPrale a la Recherche Scientifique et Technique (grant M R M 76.7.1 191) and the hstitut National de la Sunti et de la Recherche Midicale (grant 77.84) for financial support. We thank Drs A. Spassky, J. Yaniv and N . Kjeldgaard for helpful discussions, and Drs A.-L. Haenni and A. Ullmann for constructive criticisms of the manuscript.
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111 14. Meagher, R. B., Tait, R. C., Betlach, M. & Boyer, H. W . (1977) Cell 10, 521 -536. 15. Ogata, R. &Gilbert, W. (1977) Proc. NatlAcad. Sci. U.S.A. 74, 4973 -4976. 16. Boyer, H . W. & Roulland-Dussoix, D. (1969) J . Mol. Biol. 41, 459 - 472. 17. Novick, R. P. & Maas, W. K. (1961) J . Bacteriol. 81, 236-240. 18. Miller, J. H . (1972) Experiments in Molecular Genrtic,s, Cold Spring Harbour Laboratory, New York. 19. KapuScinski, J. & Skoczylas, B. (1977) Anal. Biochem. 83, 252 - 257. 20. Lowry, 0. H., Rosebrough, N . J., Farr, A. L. & Randall, R. J. (1951) J . Biol. Chem. 193, 265-275. 21. Meyers, J. A., Sanchez, D., Elwell, L. P. & Falkow, S. (1976) J . Bacteriol. 127, 1529- 1533. 22. Laemmli, U. K. (1970) Nature (Lond.) 227, 680-685. 23. Sober, H. A. (ed.) (1968) Handbook qf Biochemistry: Selected Data ,for Molecular Biology, section C, pp. C1 -C193, The Chemical Rubber Co, Cleveland. 24. Burgess, R. R. (1976) in R N A Polymerase (Losick, R. C? Chamberlin, M., eds) pp. 69- 100, Cold Spring Harbour Laboratory, New York. 25. Ovchinnikov, Y. A,, Lipkin, V. M., Modyanov, N . N., Chertov, 0. Y . & Smirnov, Y. V. (1977) FEBS Lett. 76, 108- 11 1. 26. Beyreuther, K. (1978) Nature (Lond.) 274, 767. 27. Titani, K., Koide, A,, Hermann, J., Ericsson, L. H., Kumar, S., Wade, R. D., Walsh, K. A., Neurath, H. & Fischer, E. H. (1977) Proc. Nut1 Acad. Sci. U.S.A. 74, 4762-4766. 28. Zabin, I. & Fowler, A. V. (1970) in The Lactose Operon (Beckwith, J. R. & Zipser, D., eds) pp. 27-47, Cold Spring Harbour Laboratory, New York. 29. Bonner, W. M . & Laskey, R. A. (1974) Eur. J . Biochem. 46, 83-88. 30. Laskey, R. A. & Mills, A. D. (1975) Eur. J . Biochem. 56, 335 - 341. 31. Tanaka, T. & Weisblum, B. (1975) J . Bacteriol. 121, 354-362. 32. Burgess, R. R. & Jendrisak, J. J. (1975) Biochemistry, 14,46344638. 33. Means, G. E. & Feeney, R . E. (1971) in Clzemical Modr/i'cation of Proteins, p. 216, Holden-Day, San Francisco. 34. Rigomier, D. & Lubochinsky, B. (1974) Ann. Microhiol. (Paris) 12SB, 295 - 303. 35 Zolotor, L. & Engler, R. (1967) Biochim. Biophys. Acta, 145, 52 - 59. 36. Gilbert, W., Gralla, J., Majors, J. Sr Maxam, A. (1975) in Protein Ligand Interactions (Sund, H . & Blauer, G., eds) pp. 193-210, de Gruyter, Berlin. 37. Von Hippel, P. H., Revzin, A,, Gross, C. A. & Wang, A. C. (1975) in Protein Ligand Interactions (Sund, H. & Blauer, G., eds) pp. 270-288, de Gruyter, Berlin. 38. Jacquet, M., Cukier-Kahn, R., Pla, J. & Gros, F. (1971) Biochem. Biophys. Res. Commun. 45, 1597- 1607. 39 Spassky, A. & Buc, H. C. (1977) Eur. J . Biochem. 81, 79-90. 40 Farabaugh, P. J. (1978) Nature (Lond.) 274, 765-769. 41, Bourgeois, S. & Pfahl, M. (1976) Adv. Protein Cliem. 30, 1-99.
S. Busby, A. Kolb, and H. Buc*, Departement de Biologie Moleculaire, Institut Pasteur, 25/28 Rue du Docteur-Roux, F-75724 Paris-Cedex-15, France
* To whom correspondence should be addressed.
