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The promoter-operator region of the Lac operon of Escherichia coli
J. Mol. Biol (1968) 38, 413-420
The Promoter-Operator Region of the Zac Operon of Escherichia coli JEFFREY H. MILLER,
KARIN
IPPEN, JOHN G. ScAIFE’f’ AND JON R. BECKWITH
Department of Bacteriology and Immunology, Harvard Medical School, Boston, Massachusetts 02115, U.S.A. (Received 15 August 1968) Deletion mapping of the Zuc operon confirms the order i-p-o-z-y-a. The promoter and operator sites are substantially distinct from each other. The promoter region is shown to be essential for the expression of the Zuc operon; deletion of this region severely affects lac activity. The combined promoter-operator region between the i and z genes is much smaller than the average gene.
1. Introduction The luc (lactose) operon of Escherichia coli consists of three structural genes, z (for pgalactosidase), y (for permease) and a (for thiogalactoside-transacetylase) and their closely linked controlling elements (Jacob & Monod, 1961; Beckwith, 1967). The first step in the expression of the operon is the initiation of transcription of these genes into an mRNA copy. This transcription, which is initiated at a site termed the promoter (for definition of terms, see Epstein & Beckwith, 1968), is prevented by the interaction of the luc repressor (the i gene product) with the lac operator site on the DNA (Gilbert & Miiller-Hill, 1967; Riggs, Bourgeois, Newby & Cohn, 1968). The mechanism by which the repressor blocks Zac operon mRNA synthesis is not yet known. However, important to an understanding of this mechanism is a knowledge of the location, properties and relationship of the luc operator (0) and promoter (p) sites. We have recently presented evidence that the order of elements in the luc operon is i-p-o-z-y-a (Ippen, Miller, Scaife & Beckwith, 1968). In this paper, we describe further evidence for this order and for the essentiality of the lac promoter region for operon expression. In addition, we show that the operator and promoter regions are distinct from each other and that they occupy a relatively small region between the i gene and the z gene.
2. Materials and Methods (a) Bacterial
straim
and
markers
The proC (proline) marker has been described (Signer & Beckwith, 1966); the galE’ mutant, PL132, is that used by Malamy (1966); the F-lac-proA, B episome was isolated by E. R. Signer. The transposition strains, lysogenic for the two $80 dlac’s (Signer t Beckwith, 1966), carry a deletion (Xl 11) of the Zuc-proA, B region. This deletion strain, CA150, and the Zac markers, ic,,,,, O&, Oi,,, U118 and X90, were obtained from F. Jacob. The ymutant, is an amber described previously (Beckwith t Signer, 1966). The four mutants, Ll, LS, L29 and L37, have been described (Scaife t Beckwith, 1966; Ippen et al., 1968). t Present address : Medical Research Council Microbial Genetics Reseamh Unit, Department of Molecular Biology, The University, King’s Building, West Mains Road, Edinburgh 9, Scotland. 28
413
414
J. H. MILLER
ET AL.
(b) Chemkak 5-Chloro-4-bromo-3-indolyl-b-n-galectoside was purchased from the Cycle Chemical Corporation. Phenyl-/3-n-galactoside was purchased from the Sigma Chemical Company.
The media used are M63 LB broth, which contains of water, and F top agar Galactosidase was assayed
(c) Media und assays minimal (Pardee, Jacob & Monod, 1959), B broth (Signer, 1966), 10 g Bacto-tryptone, 5 g yeast extract, and 10 g NaCl in 1 liter which contains 7.5 g agar and 8 g NaCl in 1 liter of water. fiaccording to Pardee et al. (1969).
(d) Three factor croaaea Hfr and F- strains were grown to 2 x lo* cells/ml. in LB broth, mixed in equal volumes and allowed to mate for 60 min at which time the mating was interrupted in the dilution tubes by vigorous agitation on a Vortex mixer for 60 sec. From a IO-fold dilution, 0.1 ml. was put into 2.5 ml. of F top agar and the soft agar poured onto melibiose minimal plates for the nzel +po + recombinants. It will be recalled that melibiose can be used to select for y gene activity at 42°C (Beckwith, 1963). In addition, 0.1 ml. of a 10M3 dilutionwas plated on glucose minimal agar for the pro + recombinants. (e) Iaolution of Tlr mutants Tl’ mutants from type I and type II strains (see Fig. 1) were selected using the colioin V and B lysates and QSOvlysates as described previously (Beckwith & Signer, 1966). We shall present full details on such selections in a subsequent publication (Miller, Reznikoff, Signer & Beckwith, manuscript in preparation). (f) Deletion mapping In all cases, an F-luc-proA, B episome carrying the lac mutation in question is introduced into the Tlr deletion-&-pro deletion strain by selecting for the proline marker. Many independent cultures of these diploid strains were grown in LB broth or B broth with glucose to 2 x lo* cells/ml. and appropriate dilutions plated on the relevant medium. lac constitutive mutants were mapped by either counting the inducible colonies formed on XGt indicator medium (spreading a few thousand bacteria per plate, see Ippen et al., 1968), or by direct selection of inducible recombinants. Such recombinants are selected in galepimerase negative derivatives of the Tl’-lac deletion strains infected with F&c+ (OO)pro+ . The diploid strains are plated on minimal glucose medium containing 0.03% PG so as to give up to 100,000 colonies per plate. The constitutive parents produce fl-gdactosidaae, hydrolysing the non-inducing phenyl galaotoside and yielding galactose. The galactose kills the strain since it is gal-epimeraae negative. Inducible recombinants have no ,%galactosidase and therefore survive (Davies & Jacob, 1968). PC-resistant colonies were streaked on XG plates to determine if they were inducible for kzc or not. Colonies thought to behave aa wild-type recombinants were grown up and verified by fl-galactosidase assay. With X8608, the frequency of PG-resistant colonies which were not recombinants was approximately 10T4 and with X8554 and X8565, approximately 10v6. We have no explanation for this difference. The non-recombinant PG-resistants were either g&lactose-resistant mutants or Zuc- mutants (see Malamy, 1966).
3. Results (a) Mapping of the p region We have described four (p- ) mutants which lower the maximum level of & operon expression (Scaife & Beckwith, 1966 ; Ippen et al., 1968). Three of these, L8, L29 and L37, appear to have normal operators and i gene activity, while a fourth, Ll, has a normal operator, but impaired i gene activity (Table 1). In addition, we have shown t Abbreviations gala&&de.
used : XG,
5-chloro-4-bromo-3-indolyl-B-D-gelectoside;
PG, phenyl-p-n-
PROMOTER-OPERATOR
REGION TABLE
415
OF I;AC
1
/3-Galuctosidase level of mutants of the Lc promoter region Induced
L8, L29, L37 Ll X8605 Wild type
6 2 0.4 100
Uninduced Diploid with Haploid F’lac (z-i’) o-02 9.1 -
o-02 l-5 0.4 0.1
Enzyme levels are reported as y0 of t,he folly induced wild-type level. Both Ll and X8606 behave as i- mutants. The Oc character of X8605 may be due to partial deletion of the operator.
TABLE 2 Recombination frequencies in the lac operon Hfr Ll
L8
L37
L29
U118
x90 .53 L8
FLl L8 L37 L29 U118 Ll L8 L37 L29 U118 Ll L8 L37 U118 Ll L8 L29 U118 Ll L8 L37 L29 U118 L8 0:s
o/0 recombinants/proC
+
< 0~0001 <0~0002
me2 +pro + reoombinants were scored for all crosses except the last three. For the cross between X90 and Ul 18, ZUC+pro + recombinents were scored. The recombination distance between ii and L8 was determined in a cross between CA150 (Hfr Hayes kc-iizi Sms) and X8001 (F-Zoc~apr~C-Srn~). pro+ recombmants were selected on XG plates. In this cross, the constitutive recombinents detected on XC plates were either of genotype i 3 Zuc+ or i 3 lacm. The latter can be distinguished from the former due to the p - levels of j3-galactosidase. The frequency of recombination between L8 and 00 is determined in a cross between CA8003 (Hfr Hayes ZacmSms) and X8030 (F-lac-i~01C6y;szo3ZNoC-Smr). meZ+ recombinants are detected and scored to determine whether they are of genotype OOp+z+y + or 0 +p +z +y +. The proportion of 0 * amongst the total number was 2.5%. This frequency represents the LS-0:s distance relative to the distance from L8 to the y - mutant. This latter distance should not be much more than 3.4 map units, considering the relative size of the z gene and y gene products (Brown et al., 1966; Steers et al., 1965; Jones & Kennedy, 1968). Thus, the L&O& distance by this estimate is 0.08 map unit.
416
J. H. MILLER
ET
AL.
that the mutants, L8, L29 and L37, are point mutations (Arditti, Miller, Scaife & Beckwith, 1968; Arditti, Soaife & Beckwith, 1968). Reciprocal three-factor crosses using the bacterial conjugation system have been used to determine the mapping relationship between the p- mutants. The proC locus, which is closely linked (about 95%) to the lac region, but transferred after it by Hfr Hayes, can be used as the selected marker in such crosses. The results of these crosses are presented in Table 2. From these data, we can draw the following conclusions: (1) L8 and L37 probably map at the same site. The total map distance of the z gene, which must, be approximately 3700 base pairs long (Steers, Craven, Anfinsen & Bethune, 1965; Brown, Koorajian, Katze & Zabin, 1966) is about 2.7%. The absence of recombination between L8 and L37 at l/10,000 this frequency indicates either identity of the two sites, or extremely close proximity. (2) L29 maps at a different site from L8-L37. (3) The mutant, Ll, is a deletion covering L29 and the L8-L37 sites. (b) Relative positions of operator and promoter In addition to these three-factor crosses, we have extended the deletion mapping studies previously reported. The deletions of the lac controlling elements are isolated in two different strains of E. wli in which the luc operon is transposed from its normal position to a site on the chromosome adjacent to the trp (tryptophan) operon (Fig. 1). Iac ott80 480 genes, a y TYP 1
z
(“’
f'P o p I Ott80 (‘I’
TI A BCDEO
r
X8605
lac att 80 TYPE n
ipo z c I ;I (1 I I II / II I /I/ I /I II /’I I ’ /
frp 5580 ott a0 T, genes
ya r
X8504 X8507 X85.55 X8554 X8508
A BCDEO
----. I ___---_
FICA 1. Tlr - Deletions of the &zc operon in 480 dZac lysogens. Some of the d&x for these deletions is presented in Ippen et al. (1908).
The lac operon, in these strains, is incorporated into the chromosome as part of the (680 dlac transducing phage (Signer & Beckwith, 1966). The two strains differ in that the lccc operons have opposite orientations relative to the trp operon (Beckwith Q Signer, 1966). Located between the lac and trp operons in these strains is a locus determining sensitivity to the bacteriophage Tl. Many of the mutants selected for resistance to Tl are deletions extending into either the structural genes of the ~UC operon, the controlling elements of the luc operon or the i gene (Beckwith, Signer t Epstein, 1966; Ippen et al., 1968). The deletions from a type II strain described here were isolated as TI’-lacmutants which still recombine with the early i gene mutation ii,,,, (Davies & Jacob, 1968). Of these deletions, one, X8554, cuts into the oontroting element region of the lac operon. This deletion strain retains an intact i gene; when an episome of
PROMOTER-OPERATOR
REGION TABLE
417
OF LAG’
3
Deletion mapping of the promoter-operator region X8504
X8555
Deletions X8654
%I,
+ + 0.79 0.52
0.065 + 0.086 0.0022
Ok
0.23 (0.26)
0.0015
oandp Mutants L8 L29 L37
Some of these data and mapping frequencies represent the percentage diploids (see Materials and Methods).
X8508
X8605
0.087 + 0.086 < 0.00008
0 0 0 < 0.00008
< 0*0001 < 0*0001 0.005
0~00017
< 0~00007
0.02
techniques have been published (Ippen et aE., 1968). The of recombinants colonies recovered from stable F-Zac-pro Recombination between the pairs O&/X8605; O’&,,/X8605
end Oi5/X8504 (shown in parenthesis) w&s observed on XG indicator medium. Recombinants from the remaining Oc/deletion pairs were selected by the PG technique (see Materials and Methods). Recombinants from the p-/deletion pairs were observed on eosin-methylene bluelactose indicator medium.
character F&c (i-o +Z+ ) is introduced into this strain, the episomal lac operon is fully repressed. In addition, X8554 recombines with the promoter mutants, Ll, L8, L29 and L37 (Table 3). We have reported the failure to detect any i +o + recombinants between this deletion and two 0” mutants, O&, and O&,, (Ippen et al., 1968). Using a more sensitive technique (Davies & Jacob, 1968) permitting direct selection of such recombinants, we again find no recombination of X8554 with O&, but an extremely low frequency with O,,. Particularly since the order of markers in this region is supposed to be p-O&-O&,-z (Davies & Jacob, 1968; see X8605 and X8555 frequencies, Table 3), we wonder whether the apparent 0+ recombinants (3 were found) between O&, and X8554 may be either revertants or may reflect some abnormal property of this marker. In any event, the deletion, X8554, appears to remove all or most of the luc operator but does not cut into the promoter. A second deletion, X8555, does not recombine with any z- mutations, but does give a low frequency of recombination with O& and ,O&. This deletion cuts very close to the operator or removes a terminal portion of it. A third deletion into this region, X8605, was isolated from a type I strain. This deletion was isolated also as a Tlr derivative, but this time on the sensitive XG indicator plates which reveal mutants with even slightly constitutive levels of the lac enzymes. The mutant was picked up as a weakly constitutive strain on such plates. The level of ,Sgalactosidase, which is not superinducible, is about 0.5% of the maximal level of lac operon expression. In this strain the i gene is completely deleted and the deletion fails to recombine with the p- mutants, L29 and L8 (Table 3). However, this deletion does recombine with both Oq, and O&. In addition, we have shown that the operator is, at least, partially intact, since introduction of an F&c (i+ ) episome into this strain results in a partial repression of the low level of expression of the chromosomally located z gene (Table 1). (c) Map distances in the lac operon A series of three-factor crosses were carried out with various mutants of the i gene, the o and p regions and the z and y genes. The recombination frequencies are presented
418
ET AL.
J. H. MILLER I
P. 0
I
I ,’
,; /’
I I’
Y
U118 ‘\ ‘\
x90
‘\\
A29 L8 IA
‘\ Oh7 2:
FIQ. 2. Map distances in the Zac operon. This map is drawn roughly to scale on the basis of the map distances presented here and on the size of the i gene (Riggs & Bourgeois, 1968; Gilbert & Miilier-Hill, personal communication), and z gene products (Brown et al., 1966; Steers et al., 1965). U118 and X90 are the most proximal and most distal well-characterized z- mutations, respectively (Newton, Beckwith, Zipser t Brenner, 1966). The order of L29 and L8-L37 is not known.
in Table 2. The various frequencies all point to the same scale of distances in the lac operon (Fig. 2). 4. Discussion We have proposed that there is a p region lying between the operator and the i gene which determines the initiation of transcription of the lac operon (Ippen et al., 1968). One of the steps in this initiation process must be the binding of the transcription enzyme, presumably RNA polymerase, to a site in this region. Although there may be other steps involved (e.g. Imamoto, 1968; Baker t Yanofsky, 1968), one possible role for the promoter site is to act as such a binding site. The following discussion covers the evidence for the location and properties of the p region. (a) Eseentiality of the promoter region It appears very likely that the ‘p-” mutants described here define an initiation site in lac operon expression. However, the possibility remains that the mutants, L8, L29, L37 and Ll, do not lie in an essential region of the operon, but rather, in some undefined way, generate interference with operon expression. The properties of the two deletion strains, Ll and X8605, make this explanation unlikely. Both of the deletions remove at least a substantial portion of the p region, while leaving the operator nearly or entirely intact. In both cases, the expression of the l.u~ operon is severely impaired (50 times for Ll and 250 times for X8605) by the removal of the promoter region. These results indicate that the region defined by the p- mutants is essential to lac operon expression. (b) Relative position of operator and promoter The deletion mapping data presented here con&m the order previously proposed, i-p-o-z-y-a. In addition, these data allow us to explain the properties of the pmutant, Ll. This mutant is a deletion covering a substantial portion of the promoter and extending into a terminal segment of the i gene. (c) Distinction
of o and p
The results presented here, in conjunction with those described elsewhere, indicate that the operator and promoter regions are substantially distinct from each other.
PROMOTER-OPERATOR
REGION
OF LAC
419
That is, the sequence of bases comprising the operator region serves only as a binding site for the repressor, while the promoter region determines only the initiation of operon expression. First, promoter point mutants, and more importantly, a deletion of the promoter region, Ll, have no or little effect on the binding of repressor. In diploid strains this latter mutant is strongly repressiblei. Second, 0” mutants, including ones characterized as deletions (Davies $ Jacob, 1968), have no effect on the maximum level of lac operon expression. These results do not eliminate the possibility that there is some overlap at the juncture of o and p, but do suggest that, for the most part,, these regions are distinct. Despite deletion of the p region in Ll and X8605, there still remains a low, but significant level of luc operon expression in these strains. This low level may be due to incomplete deletion of the promoter region : or, in the case of X8605, the low level may be due to the particular properties of the Tl’ deletion. It is possible that X8605 fuses lac, through its promoter to some other gene or operon which is functioning at a very low level. An alternative explanation for this property of Ll and X8605 is that the intact operator in these strains can itself serve as a weak initiation site. (d) Size of the operatorq-order
region
It is clear that one cannot obtain a precise estimate of true distances from recombination frequencies (Yanofsky, Carlton, Guest, Helinski & Henning, 1964). Marker effects on recombination consistently interfere with the accuracy of such data. A good example of this complication is the severe effect of the mutant Ll on the recombination frequencies. Ll, a deletion, which is as close to the mutant U118 or closer than L8, gives an approximately sevenfold higher recombination frequency with U118. However, since Ll is a deletion, effects on recombination frequencies are not unexpected. The rest of the date, based mainly on recombination between point mutants is consistent with the map presented in Figure 2. The mapping data indicate that the region between the i gene and the z gene is much smaller than the average gene. On the basis of a ratio of 2-7 map units per 3700 base pairs for the z gene (Table 2)) we would estimate from the series of recombination frequencies that the combined p-o region is on the order of 100 base-pairs long. Again, these data only provide a very rough estimate for the size of this region for the reasons cited above. This work was supported by grant no. One of us (J.R.B.) is supported by a Institutes of Health. Also one of us (J. Health training grant to the Department University, Cambridge, Massachusetts.
GM-13017 from the National Institutes of Health. Career Development Award from the National H. M.) is supported by a National Institutes of of Biochemistry and Molecular Biology, Harvard
REFERENCES Arditti, R. R., Miller, J. H., Scaife, J. & Beckwith, J. (1968). In MolecuZ~ Genetics, Symposium of the Gesellschaft Deutscher Naturforsoher und &zter, in the press. Arditti, R. R., Scaife, J. & Beckwith, J. R. (1968). J. Mol. Biol. 38,421. Baker, R. F. & Yanofsky, C. (1968). Proc. Nat. Acad. SC&, W&L. 60, 313. Beckwith, J. R. (1963). Biochim. biophys. Acta, 76, 162. Beckwith, J. R. (1967). Science, 156, 697. Beckwith, J. R. & Signer, E. R. (1966). J. Mol. Biol. 19, 254.
A
t Admittedly, all 4 p- mutants have repressed levels higher than expected from the normal 1OOO:l induction ratio (Table 1). However, the constancy of this level amongst all 4 mutants suggests that this may be an unrepressible bass1 level below which it is impossible to go.
420
J. H. MILLER
ET
AL.
Beckwith, J. R., Signer, E. R. & Epstein, W. (1966). Cold&n-. Harb. Symp. Quant. Biol. 31, 393. Brown, J. L., Koorajian, S., Katze, J. & Zabin, I. (1966). J. Biol. Chem. 241, 2826. Davies, J. & Jacob, F. (1968). J. Mol. Biol. 36, 413. Epstein, W. & Beckwith, J. R. (1968). Ann. Rev. Biochem., 37, 411. Gilbert, W. & Miiller-Hill, B. (1967). Proc. Nat. Acad. Sci., Wash. 58, 2415. Imamoto, F. (1968). Proc. Nut. Acad. Sci., Wash. 60, 305. Ippen, K., Miller, J. H., Scaife, J. G. & Beokwith, J. R. (1968). Nature, 217, 825. Jacob, F. & Monod, J. (1961). J. Mol. Biol. 3, 318. Jones, T. H. D. & Kennedy, E. P. (1968). Fed. Proc. 27, 644. Malamy M. (1966). Cold Spr. Harb. Symp. Quant. Biol. 31, 189. Newton, W. A., Beckwith, J. R., Zipser, D. & Brenner, S. (1965). J. Mol. BioZ. 14, 290. Pardee, A. B., Jacob, F. & Monod, J. (1959). J. Mol. BioZ. 1, 165. Riggs, A. D. & Bourgeois, S. (1968). J. MoZ. BioZ. 34, 361. Riggs, A. D., Bourgeois, S., Newby, R. F. & Cohn, M. (1968). J. Mol. BioZ. 34, 365. Scaife, J. G. & Beckwith, J. R. (1966). Cold Spr. Harb. Symp. Quant. BioZ. 31, 403. Signer, E. R. (1966). J. Mol. BioZ. 15, 243. Signer, E. R. & Beckwith, J. R. (1966). J. Mol. BioE. 22, 33. Steers, E., Craven, G. R., Anflnsen, C. B. & Bethune, J. L. (1965). J. BioZ. Chem. 240,2478. Yanofsky, C., Carlton, B. C., Guest, J. R., Helinski, D. R. & Henning, U. (1964). Proc. Nat. Acad. Sci., Wash. 51, 266.
The Promoter-Operator Region of the Zac Operon of Escherichia coli JEFFREY H. MILLER,
KARIN
IPPEN, JOHN G. ScAIFE’f’ AND JON R. BECKWITH
Department of Bacteriology and Immunology, Harvard Medical School, Boston, Massachusetts 02115, U.S.A. (Received 15 August 1968) Deletion mapping of the Zuc operon confirms the order i-p-o-z-y-a. The promoter and operator sites are substantially distinct from each other. The promoter region is shown to be essential for the expression of the Zuc operon; deletion of this region severely affects lac activity. The combined promoter-operator region between the i and z genes is much smaller than the average gene.
1. Introduction The luc (lactose) operon of Escherichia coli consists of three structural genes, z (for pgalactosidase), y (for permease) and a (for thiogalactoside-transacetylase) and their closely linked controlling elements (Jacob & Monod, 1961; Beckwith, 1967). The first step in the expression of the operon is the initiation of transcription of these genes into an mRNA copy. This transcription, which is initiated at a site termed the promoter (for definition of terms, see Epstein & Beckwith, 1968), is prevented by the interaction of the luc repressor (the i gene product) with the lac operator site on the DNA (Gilbert & Miiller-Hill, 1967; Riggs, Bourgeois, Newby & Cohn, 1968). The mechanism by which the repressor blocks Zac operon mRNA synthesis is not yet known. However, important to an understanding of this mechanism is a knowledge of the location, properties and relationship of the luc operator (0) and promoter (p) sites. We have recently presented evidence that the order of elements in the luc operon is i-p-o-z-y-a (Ippen, Miller, Scaife & Beckwith, 1968). In this paper, we describe further evidence for this order and for the essentiality of the lac promoter region for operon expression. In addition, we show that the operator and promoter regions are distinct from each other and that they occupy a relatively small region between the i gene and the z gene.
2. Materials and Methods (a) Bacterial
straim
and
markers
The proC (proline) marker has been described (Signer & Beckwith, 1966); the galE’ mutant, PL132, is that used by Malamy (1966); the F-lac-proA, B episome was isolated by E. R. Signer. The transposition strains, lysogenic for the two $80 dlac’s (Signer t Beckwith, 1966), carry a deletion (Xl 11) of the Zuc-proA, B region. This deletion strain, CA150, and the Zac markers, ic,,,,, O&, Oi,,, U118 and X90, were obtained from F. Jacob. The ymutant, is an amber described previously (Beckwith t Signer, 1966). The four mutants, Ll, LS, L29 and L37, have been described (Scaife t Beckwith, 1966; Ippen et al., 1968). t Present address : Medical Research Council Microbial Genetics Reseamh Unit, Department of Molecular Biology, The University, King’s Building, West Mains Road, Edinburgh 9, Scotland. 28
413
414
J. H. MILLER
ET AL.
(b) Chemkak 5-Chloro-4-bromo-3-indolyl-b-n-galectoside was purchased from the Cycle Chemical Corporation. Phenyl-/3-n-galactoside was purchased from the Sigma Chemical Company.
The media used are M63 LB broth, which contains of water, and F top agar Galactosidase was assayed
(c) Media und assays minimal (Pardee, Jacob & Monod, 1959), B broth (Signer, 1966), 10 g Bacto-tryptone, 5 g yeast extract, and 10 g NaCl in 1 liter which contains 7.5 g agar and 8 g NaCl in 1 liter of water. fiaccording to Pardee et al. (1969).
(d) Three factor croaaea Hfr and F- strains were grown to 2 x lo* cells/ml. in LB broth, mixed in equal volumes and allowed to mate for 60 min at which time the mating was interrupted in the dilution tubes by vigorous agitation on a Vortex mixer for 60 sec. From a IO-fold dilution, 0.1 ml. was put into 2.5 ml. of F top agar and the soft agar poured onto melibiose minimal plates for the nzel +po + recombinants. It will be recalled that melibiose can be used to select for y gene activity at 42°C (Beckwith, 1963). In addition, 0.1 ml. of a 10M3 dilutionwas plated on glucose minimal agar for the pro + recombinants. (e) Iaolution of Tlr mutants Tl’ mutants from type I and type II strains (see Fig. 1) were selected using the colioin V and B lysates and QSOvlysates as described previously (Beckwith & Signer, 1966). We shall present full details on such selections in a subsequent publication (Miller, Reznikoff, Signer & Beckwith, manuscript in preparation). (f) Deletion mapping In all cases, an F-luc-proA, B episome carrying the lac mutation in question is introduced into the Tlr deletion-&-pro deletion strain by selecting for the proline marker. Many independent cultures of these diploid strains were grown in LB broth or B broth with glucose to 2 x lo* cells/ml. and appropriate dilutions plated on the relevant medium. lac constitutive mutants were mapped by either counting the inducible colonies formed on XGt indicator medium (spreading a few thousand bacteria per plate, see Ippen et al., 1968), or by direct selection of inducible recombinants. Such recombinants are selected in galepimerase negative derivatives of the Tl’-lac deletion strains infected with F&c+ (OO)pro+ . The diploid strains are plated on minimal glucose medium containing 0.03% PG so as to give up to 100,000 colonies per plate. The constitutive parents produce fl-gdactosidaae, hydrolysing the non-inducing phenyl galaotoside and yielding galactose. The galactose kills the strain since it is gal-epimeraae negative. Inducible recombinants have no ,%galactosidase and therefore survive (Davies & Jacob, 1968). PC-resistant colonies were streaked on XG plates to determine if they were inducible for kzc or not. Colonies thought to behave aa wild-type recombinants were grown up and verified by fl-galactosidase assay. With X8608, the frequency of PG-resistant colonies which were not recombinants was approximately 10T4 and with X8554 and X8565, approximately 10v6. We have no explanation for this difference. The non-recombinant PG-resistants were either g&lactose-resistant mutants or Zuc- mutants (see Malamy, 1966).
3. Results (a) Mapping of the p region We have described four (p- ) mutants which lower the maximum level of & operon expression (Scaife & Beckwith, 1966 ; Ippen et al., 1968). Three of these, L8, L29 and L37, appear to have normal operators and i gene activity, while a fourth, Ll, has a normal operator, but impaired i gene activity (Table 1). In addition, we have shown t Abbreviations gala&&de.
used : XG,
5-chloro-4-bromo-3-indolyl-B-D-gelectoside;
PG, phenyl-p-n-
PROMOTER-OPERATOR
REGION TABLE
415
OF I;AC
1
/3-Galuctosidase level of mutants of the Lc promoter region Induced
L8, L29, L37 Ll X8605 Wild type
6 2 0.4 100
Uninduced Diploid with Haploid F’lac (z-i’) o-02 9.1 -
o-02 l-5 0.4 0.1
Enzyme levels are reported as y0 of t,he folly induced wild-type level. Both Ll and X8606 behave as i- mutants. The Oc character of X8605 may be due to partial deletion of the operator.
TABLE 2 Recombination frequencies in the lac operon Hfr Ll
L8
L37
L29
U118
x90 .53 L8
FLl L8 L37 L29 U118 Ll L8 L37 L29 U118 Ll L8 L37 U118 Ll L8 L29 U118 Ll L8 L37 L29 U118 L8 0:s
o/0 recombinants/proC
+
< 0~0001 <0~0002
me2 +pro + reoombinants were scored for all crosses except the last three. For the cross between X90 and Ul 18, ZUC+pro + recombinents were scored. The recombination distance between ii and L8 was determined in a cross between CA150 (Hfr Hayes kc-iizi Sms) and X8001 (F-Zoc~apr~C-Srn~). pro+ recombmants were selected on XG plates. In this cross, the constitutive recombinents detected on XC plates were either of genotype i 3 Zuc+ or i 3 lacm. The latter can be distinguished from the former due to the p - levels of j3-galactosidase. The frequency of recombination between L8 and 00 is determined in a cross between CA8003 (Hfr Hayes ZacmSms) and X8030 (F-lac-i~01C6y;szo3ZNoC-Smr). meZ+ recombinants are detected and scored to determine whether they are of genotype OOp+z+y + or 0 +p +z +y +. The proportion of 0 * amongst the total number was 2.5%. This frequency represents the LS-0:s distance relative to the distance from L8 to the y - mutant. This latter distance should not be much more than 3.4 map units, considering the relative size of the z gene and y gene products (Brown et al., 1966; Steers et al., 1965; Jones & Kennedy, 1968). Thus, the L&O& distance by this estimate is 0.08 map unit.
416
J. H. MILLER
ET
AL.
that the mutants, L8, L29 and L37, are point mutations (Arditti, Miller, Scaife & Beckwith, 1968; Arditti, Soaife & Beckwith, 1968). Reciprocal three-factor crosses using the bacterial conjugation system have been used to determine the mapping relationship between the p- mutants. The proC locus, which is closely linked (about 95%) to the lac region, but transferred after it by Hfr Hayes, can be used as the selected marker in such crosses. The results of these crosses are presented in Table 2. From these data, we can draw the following conclusions: (1) L8 and L37 probably map at the same site. The total map distance of the z gene, which must, be approximately 3700 base pairs long (Steers, Craven, Anfinsen & Bethune, 1965; Brown, Koorajian, Katze & Zabin, 1966) is about 2.7%. The absence of recombination between L8 and L37 at l/10,000 this frequency indicates either identity of the two sites, or extremely close proximity. (2) L29 maps at a different site from L8-L37. (3) The mutant, Ll, is a deletion covering L29 and the L8-L37 sites. (b) Relative positions of operator and promoter In addition to these three-factor crosses, we have extended the deletion mapping studies previously reported. The deletions of the lac controlling elements are isolated in two different strains of E. wli in which the luc operon is transposed from its normal position to a site on the chromosome adjacent to the trp (tryptophan) operon (Fig. 1). Iac ott80 480 genes, a y TYP 1
z
(“’
f'P o p I Ott80 (‘I’
TI A BCDEO
r
X8605
lac att 80 TYPE n
ipo z c I ;I (1 I I II / II I /I/ I /I II /’I I ’ /
frp 5580 ott a0 T, genes
ya r
X8504 X8507 X85.55 X8554 X8508
A BCDEO
----. I ___---_
FICA 1. Tlr - Deletions of the &zc operon in 480 dZac lysogens. Some of the d&x for these deletions is presented in Ippen et al. (1908).
The lac operon, in these strains, is incorporated into the chromosome as part of the (680 dlac transducing phage (Signer & Beckwith, 1966). The two strains differ in that the lccc operons have opposite orientations relative to the trp operon (Beckwith Q Signer, 1966). Located between the lac and trp operons in these strains is a locus determining sensitivity to the bacteriophage Tl. Many of the mutants selected for resistance to Tl are deletions extending into either the structural genes of the ~UC operon, the controlling elements of the luc operon or the i gene (Beckwith, Signer t Epstein, 1966; Ippen et al., 1968). The deletions from a type II strain described here were isolated as TI’-lacmutants which still recombine with the early i gene mutation ii,,,, (Davies & Jacob, 1968). Of these deletions, one, X8554, cuts into the oontroting element region of the lac operon. This deletion strain retains an intact i gene; when an episome of
PROMOTER-OPERATOR
REGION TABLE
417
OF LAG’
3
Deletion mapping of the promoter-operator region X8504
X8555
Deletions X8654
%I,
+ + 0.79 0.52
0.065 + 0.086 0.0022
Ok
0.23 (0.26)
0.0015
oandp Mutants L8 L29 L37
Some of these data and mapping frequencies represent the percentage diploids (see Materials and Methods).
X8508
X8605
0.087 + 0.086 < 0.00008
0 0 0 < 0.00008
< 0*0001 < 0*0001 0.005
0~00017
< 0~00007
0.02
techniques have been published (Ippen et aE., 1968). The of recombinants colonies recovered from stable F-Zac-pro Recombination between the pairs O&/X8605; O’&,,/X8605
end Oi5/X8504 (shown in parenthesis) w&s observed on XG indicator medium. Recombinants from the remaining Oc/deletion pairs were selected by the PG technique (see Materials and Methods). Recombinants from the p-/deletion pairs were observed on eosin-methylene bluelactose indicator medium.
character F&c (i-o +Z+ ) is introduced into this strain, the episomal lac operon is fully repressed. In addition, X8554 recombines with the promoter mutants, Ll, L8, L29 and L37 (Table 3). We have reported the failure to detect any i +o + recombinants between this deletion and two 0” mutants, O&, and O&,, (Ippen et al., 1968). Using a more sensitive technique (Davies & Jacob, 1968) permitting direct selection of such recombinants, we again find no recombination of X8554 with O&, but an extremely low frequency with O,,. Particularly since the order of markers in this region is supposed to be p-O&-O&,-z (Davies & Jacob, 1968; see X8605 and X8555 frequencies, Table 3), we wonder whether the apparent 0+ recombinants (3 were found) between O&, and X8554 may be either revertants or may reflect some abnormal property of this marker. In any event, the deletion, X8554, appears to remove all or most of the luc operator but does not cut into the promoter. A second deletion, X8555, does not recombine with any z- mutations, but does give a low frequency of recombination with O& and ,O&. This deletion cuts very close to the operator or removes a terminal portion of it. A third deletion into this region, X8605, was isolated from a type I strain. This deletion was isolated also as a Tlr derivative, but this time on the sensitive XG indicator plates which reveal mutants with even slightly constitutive levels of the lac enzymes. The mutant was picked up as a weakly constitutive strain on such plates. The level of ,Sgalactosidase, which is not superinducible, is about 0.5% of the maximal level of lac operon expression. In this strain the i gene is completely deleted and the deletion fails to recombine with the p- mutants, L29 and L8 (Table 3). However, this deletion does recombine with both Oq, and O&. In addition, we have shown that the operator is, at least, partially intact, since introduction of an F&c (i+ ) episome into this strain results in a partial repression of the low level of expression of the chromosomally located z gene (Table 1). (c) Map distances in the lac operon A series of three-factor crosses were carried out with various mutants of the i gene, the o and p regions and the z and y genes. The recombination frequencies are presented
418
ET AL.
J. H. MILLER I
P. 0
I
I ,’
,; /’
I I’
Y
U118 ‘\ ‘\
x90
‘\\
A29 L8 IA
‘\ Oh7 2:
FIQ. 2. Map distances in the Zac operon. This map is drawn roughly to scale on the basis of the map distances presented here and on the size of the i gene (Riggs & Bourgeois, 1968; Gilbert & Miilier-Hill, personal communication), and z gene products (Brown et al., 1966; Steers et al., 1965). U118 and X90 are the most proximal and most distal well-characterized z- mutations, respectively (Newton, Beckwith, Zipser t Brenner, 1966). The order of L29 and L8-L37 is not known.
in Table 2. The various frequencies all point to the same scale of distances in the lac operon (Fig. 2). 4. Discussion We have proposed that there is a p region lying between the operator and the i gene which determines the initiation of transcription of the lac operon (Ippen et al., 1968). One of the steps in this initiation process must be the binding of the transcription enzyme, presumably RNA polymerase, to a site in this region. Although there may be other steps involved (e.g. Imamoto, 1968; Baker t Yanofsky, 1968), one possible role for the promoter site is to act as such a binding site. The following discussion covers the evidence for the location and properties of the p region. (a) Eseentiality of the promoter region It appears very likely that the ‘p-” mutants described here define an initiation site in lac operon expression. However, the possibility remains that the mutants, L8, L29, L37 and Ll, do not lie in an essential region of the operon, but rather, in some undefined way, generate interference with operon expression. The properties of the two deletion strains, Ll and X8605, make this explanation unlikely. Both of the deletions remove at least a substantial portion of the p region, while leaving the operator nearly or entirely intact. In both cases, the expression of the l.u~ operon is severely impaired (50 times for Ll and 250 times for X8605) by the removal of the promoter region. These results indicate that the region defined by the p- mutants is essential to lac operon expression. (b) Relative position of operator and promoter The deletion mapping data presented here con&m the order previously proposed, i-p-o-z-y-a. In addition, these data allow us to explain the properties of the pmutant, Ll. This mutant is a deletion covering a substantial portion of the promoter and extending into a terminal segment of the i gene. (c) Distinction
of o and p
The results presented here, in conjunction with those described elsewhere, indicate that the operator and promoter regions are substantially distinct from each other.
PROMOTER-OPERATOR
REGION
OF LAC
419
That is, the sequence of bases comprising the operator region serves only as a binding site for the repressor, while the promoter region determines only the initiation of operon expression. First, promoter point mutants, and more importantly, a deletion of the promoter region, Ll, have no or little effect on the binding of repressor. In diploid strains this latter mutant is strongly repressiblei. Second, 0” mutants, including ones characterized as deletions (Davies $ Jacob, 1968), have no effect on the maximum level of lac operon expression. These results do not eliminate the possibility that there is some overlap at the juncture of o and p, but do suggest that, for the most part,, these regions are distinct. Despite deletion of the p region in Ll and X8605, there still remains a low, but significant level of luc operon expression in these strains. This low level may be due to incomplete deletion of the promoter region : or, in the case of X8605, the low level may be due to the particular properties of the Tl’ deletion. It is possible that X8605 fuses lac, through its promoter to some other gene or operon which is functioning at a very low level. An alternative explanation for this property of Ll and X8605 is that the intact operator in these strains can itself serve as a weak initiation site. (d) Size of the operatorq-order
region
It is clear that one cannot obtain a precise estimate of true distances from recombination frequencies (Yanofsky, Carlton, Guest, Helinski & Henning, 1964). Marker effects on recombination consistently interfere with the accuracy of such data. A good example of this complication is the severe effect of the mutant Ll on the recombination frequencies. Ll, a deletion, which is as close to the mutant U118 or closer than L8, gives an approximately sevenfold higher recombination frequency with U118. However, since Ll is a deletion, effects on recombination frequencies are not unexpected. The rest of the date, based mainly on recombination between point mutants is consistent with the map presented in Figure 2. The mapping data indicate that the region between the i gene and the z gene is much smaller than the average gene. On the basis of a ratio of 2-7 map units per 3700 base pairs for the z gene (Table 2)) we would estimate from the series of recombination frequencies that the combined p-o region is on the order of 100 base-pairs long. Again, these data only provide a very rough estimate for the size of this region for the reasons cited above. This work was supported by grant no. One of us (J.R.B.) is supported by a Institutes of Health. Also one of us (J. Health training grant to the Department University, Cambridge, Massachusetts.
GM-13017 from the National Institutes of Health. Career Development Award from the National H. M.) is supported by a National Institutes of of Biochemistry and Molecular Biology, Harvard
REFERENCES Arditti, R. R., Miller, J. H., Scaife, J. & Beckwith, J. (1968). In MolecuZ~ Genetics, Symposium of the Gesellschaft Deutscher Naturforsoher und &zter, in the press. Arditti, R. R., Scaife, J. & Beckwith, J. R. (1968). J. Mol. Biol. 38,421. Baker, R. F. & Yanofsky, C. (1968). Proc. Nat. Acad. SC&, W&L. 60, 313. Beckwith, J. R. (1963). Biochim. biophys. Acta, 76, 162. Beckwith, J. R. (1967). Science, 156, 697. Beckwith, J. R. & Signer, E. R. (1966). J. Mol. Biol. 19, 254.
A
t Admittedly, all 4 p- mutants have repressed levels higher than expected from the normal 1OOO:l induction ratio (Table 1). However, the constancy of this level amongst all 4 mutants suggests that this may be an unrepressible bass1 level below which it is impossible to go.
420
J. H. MILLER
ET
AL.
Beckwith, J. R., Signer, E. R. & Epstein, W. (1966). Cold&n-. Harb. Symp. Quant. Biol. 31, 393. Brown, J. L., Koorajian, S., Katze, J. & Zabin, I. (1966). J. Biol. Chem. 241, 2826. Davies, J. & Jacob, F. (1968). J. Mol. Biol. 36, 413. Epstein, W. & Beckwith, J. R. (1968). Ann. Rev. Biochem., 37, 411. Gilbert, W. & Miiller-Hill, B. (1967). Proc. Nat. Acad. Sci., Wash. 58, 2415. Imamoto, F. (1968). Proc. Nut. Acad. Sci., Wash. 60, 305. Ippen, K., Miller, J. H., Scaife, J. G. & Beokwith, J. R. (1968). Nature, 217, 825. Jacob, F. & Monod, J. (1961). J. Mol. Biol. 3, 318. Jones, T. H. D. & Kennedy, E. P. (1968). Fed. Proc. 27, 644. Malamy M. (1966). Cold Spr. Harb. Symp. Quant. Biol. 31, 189. Newton, W. A., Beckwith, J. R., Zipser, D. & Brenner, S. (1965). J. Mol. BioZ. 14, 290. Pardee, A. B., Jacob, F. & Monod, J. (1959). J. Mol. BioZ. 1, 165. Riggs, A. D. & Bourgeois, S. (1968). J. MoZ. BioZ. 34, 361. Riggs, A. D., Bourgeois, S., Newby, R. F. & Cohn, M. (1968). J. Mol. BioZ. 34, 365. Scaife, J. G. & Beckwith, J. R. (1966). Cold Spr. Harb. Symp. Quant. BioZ. 31, 403. Signer, E. R. (1966). J. Mol. BioZ. 15, 243. Signer, E. R. & Beckwith, J. R. (1966). J. Mol. BioE. 22, 33. Steers, E., Craven, G. R., Anflnsen, C. B. & Bethune, J. L. (1965). J. BioZ. Chem. 240,2478. Yanofsky, C., Carlton, B. C., Guest, J. R., Helinski, D. R. & Henning, U. (1964). Proc. Nat. Acad. Sci., Wash. 51, 266.