J. kto1. Bid. (1975) 93,311-317
LETTERS TO THE EDITOR
Genetic Mapping of a New Promoter for the ZacOperon A mutation (or group of mutations) necessary for the synthesis of increased quantities of lac mRNA has been localized within the structural gene for the Zac repressor (kzcl). This mutation is probably a new cyclic AMP-binding proteindependent promoter within the I gene. We propose that the synthesis of lac mRNA in the mutant is initiated within the I gene, and that transcriptional initiation continues to occur normally at the wild-type I promoter.
The wild-type lac promoter is dependent on cyclic AMP (adenosine 3’:s’ cyclic monophosphate) and CAP (cyclic AMP-binding protein) for the initiation of transcription of lac mRNA (de Crombrugghe et al., 1971; Eron BEBlock, 1972). It has been proposed that the lac promoter consists of two sites: one for the binding of RNA polymerase and one for the cyclic AMP-mediated binding of CAP (Beckwith et al., 1972). The binding of CAP would facilitate the initiation of transcription. The entire lac promoter and operator region has now been sequenced (Gilbert & Maxam, 1973; Maizels, 1973; W. Reznikoff, personal communication), and it should soon be possible to assign functions to specific nucleotide base sequences. There are a number of “down-promoter” mutants that map in this region (Scaife & Beckwith, 1966), but no “up-promoter” mutants (Arditti et al., 1973) although some operator mutants have an increased rate of synthesis of fl-galactosidase, apparently owing to lac mRNA of altered functional half-life (Smith & Sadler, 1971; Carlson & Smith, cited in Jobe et al., 1974). We have isolated a mutant of Escherichia coli with a new luc promoter that is CAPand cyclic AMP-dependent but a maximum of 25 times as efficient as the wild-type promoter (Bruenn & Hollingsworth, 1973). The mutant has a complex phenotype, which includes partial constitutivity for the lac operon enzymes and alteration of the kinetics of fl-galactosidase synthesis, the functional half-life of b-galactosidase mRNA, and the properties of the kzc repressor (Bruenn & Hollingsworth, 1973). The up-promoter character of the mutant is dominant in cis but not in trans, whereas the I- character is slightly dominant in trans (Bruenn & Hollingsworth, 1973). There may be more than one mutation in lac responsible for this phenotype. We have designated the responsible mutation(s) lac-741. We have mapped, as the easiest marker to follow, the I- character of lac-741 and subsequently tested recombinants for the other phenotypic properties of lac-741. We have failed to separate the phenotypic properties of lac-741 by phage Pl or Mu transduction or by recombination with a set of partial lac deletions. We find that the partial I- character conferred by kc-741 maps within the structural gene for the lac repressor and that Zuc-741does not alter the kzc operator or promoter or the operator proximal portion of the I gene. The partial constitutivity conferred by l.oc-741 was exploited in transductional and deletion mapping. Transductants were selected for growth on lactose at, 37°C (Z’) or melibiose at 42°C (Y+) and subsequently tested for constitutivity and other phenotypic proper+& conferred by bc-741. 311
pro)
CSH40 F%zcYproA+,B+~A(lac pm) CSH40 CSH34 F’lacZproA+,B+/A(lac pro) (2 = U118, an early oohre mutation; see Fig. 1) CSH34 2406B F - /!uoZ CSH46 F- A(Zuc pro) (hcI857St68h80dZucI,Z) (I = 9)
Recipient
Z+ z+ Z’
Y+ Y+ z+
Seleoted marker
or minimal characterized
mapping of h-741
1
‘I’he presence of kzc-741 in transductants picked on minimal lactose at 37°C (Z + transductants) dicated by oonstitutivity for /l-galactosidase and kzc permease. All 2~~741 transductants further properties oonferred by h-741.
El01 El01 El01
CSH36 F’lacIpoA+,B+/A(lac El01 F’lac-741/A&w,, CSH36
Donor
Phage PI tranadudnd
TABLE
0.93 (79/86) 0.36 (16/46) 0.38 (26/66)
0.60 (90/161) 0.45 (44/98) 0.67 (M/82)
Co-transduction frequenoy
melibiose at 42°C (Y + transductants) was inhad all the previously desoribed phenotypio
luc-741 luc-741 luc-741
I(C) loi?-741 I(i-)
Unseleoted marker
LETTERS
TO THE
EDITOR
313
TABLE 2 Deletion mapping of lacI741 Deletion
X8554 X8608 X8616 X8606 X8626 X8617 X8660 X8604 X8632 X8638 X8635 X8640 Haploid
parent
Diploid
Recombination frequency
ES664 ES608 ES616 ES606 ES626 ES617 ES660 ES604 ES632 ES638 E8636 E8640 El27
1*6x 10-Z 3.6 x lo-’ 4.0 x 10-4 1.0 x 10-a 1.0 x 10-4 2.3 x 1O-6
LETTERS TO THE EDITOR
Genetic Mapping of a New Promoter for the ZacOperon A mutation (or group of mutations) necessary for the synthesis of increased quantities of lac mRNA has been localized within the structural gene for the Zac repressor (kzcl). This mutation is probably a new cyclic AMP-binding proteindependent promoter within the I gene. We propose that the synthesis of lac mRNA in the mutant is initiated within the I gene, and that transcriptional initiation continues to occur normally at the wild-type I promoter.
The wild-type lac promoter is dependent on cyclic AMP (adenosine 3’:s’ cyclic monophosphate) and CAP (cyclic AMP-binding protein) for the initiation of transcription of lac mRNA (de Crombrugghe et al., 1971; Eron BEBlock, 1972). It has been proposed that the lac promoter consists of two sites: one for the binding of RNA polymerase and one for the cyclic AMP-mediated binding of CAP (Beckwith et al., 1972). The binding of CAP would facilitate the initiation of transcription. The entire lac promoter and operator region has now been sequenced (Gilbert & Maxam, 1973; Maizels, 1973; W. Reznikoff, personal communication), and it should soon be possible to assign functions to specific nucleotide base sequences. There are a number of “down-promoter” mutants that map in this region (Scaife & Beckwith, 1966), but no “up-promoter” mutants (Arditti et al., 1973) although some operator mutants have an increased rate of synthesis of fl-galactosidase, apparently owing to lac mRNA of altered functional half-life (Smith & Sadler, 1971; Carlson & Smith, cited in Jobe et al., 1974). We have isolated a mutant of Escherichia coli with a new luc promoter that is CAPand cyclic AMP-dependent but a maximum of 25 times as efficient as the wild-type promoter (Bruenn & Hollingsworth, 1973). The mutant has a complex phenotype, which includes partial constitutivity for the lac operon enzymes and alteration of the kinetics of fl-galactosidase synthesis, the functional half-life of b-galactosidase mRNA, and the properties of the kzc repressor (Bruenn & Hollingsworth, 1973). The up-promoter character of the mutant is dominant in cis but not in trans, whereas the I- character is slightly dominant in trans (Bruenn & Hollingsworth, 1973). There may be more than one mutation in lac responsible for this phenotype. We have designated the responsible mutation(s) lac-741. We have mapped, as the easiest marker to follow, the I- character of lac-741 and subsequently tested recombinants for the other phenotypic properties of lac-741. We have failed to separate the phenotypic properties of lac-741 by phage Pl or Mu transduction or by recombination with a set of partial lac deletions. We find that the partial I- character conferred by kc-741 maps within the structural gene for the lac repressor and that Zuc-741does not alter the kzc operator or promoter or the operator proximal portion of the I gene. The partial constitutivity conferred by l.oc-741 was exploited in transductional and deletion mapping. Transductants were selected for growth on lactose at, 37°C (Z’) or melibiose at 42°C (Y+) and subsequently tested for constitutivity and other phenotypic proper+& conferred by bc-741. 311
pro)
CSH40 F%zcYproA+,B+~A(lac pm) CSH40 CSH34 F’lacZproA+,B+/A(lac pro) (2 = U118, an early oohre mutation; see Fig. 1) CSH34 2406B F - /!uoZ CSH46 F- A(Zuc pro) (hcI857St68h80dZucI,Z) (I = 9)
Recipient
Z+ z+ Z’
Y+ Y+ z+
Seleoted marker
or minimal characterized
mapping of h-741
1
‘I’he presence of kzc-741 in transductants picked on minimal lactose at 37°C (Z + transductants) dicated by oonstitutivity for /l-galactosidase and kzc permease. All 2~~741 transductants further properties oonferred by h-741.
El01 El01 El01
CSH36 F’lacIpoA+,B+/A(lac El01 F’lac-741/A&w,, CSH36
Donor
Phage PI tranadudnd
TABLE
0.93 (79/86) 0.36 (16/46) 0.38 (26/66)
0.60 (90/161) 0.45 (44/98) 0.67 (M/82)
Co-transduction frequenoy
melibiose at 42°C (Y + transductants) was inhad all the previously desoribed phenotypio
luc-741 luc-741 luc-741
I(C) loi?-741 I(i-)
Unseleoted marker
LETTERS
TO THE
EDITOR
313
TABLE 2 Deletion mapping of lacI741 Deletion
X8554 X8608 X8616 X8606 X8626 X8617 X8660 X8604 X8632 X8638 X8635 X8640 Haploid
parent
Diploid
Recombination frequency
ES664 ES608 ES616 ES606 ES626 ES617 ES660 ES604 ES632 ES638 E8636 E8640 El27
1*6x 10-Z 3.6 x lo-’ 4.0 x 10-4 1.0 x 10-a 1.0 x 10-4 2.3 x 1O-6
