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dnaA box complex blocks transcribing RNA polymerase
347
Gene, 73 (1988) 347-3.54
Elsevier GEN 02763
Termination of the EsclrerMGa coii us& transwipt. The DnaA proteim/dmzA box complex blocks transcribiig RNA plymerase (Initiation of replication; oriC; mioC; galhl-expression plasmid; autore~ation;
recombinant DNA)
Christoph Schaefer and Walter Messer Max-Planck-Institut fiir Molekulare Gene&, Berlin (F.R.G.) Received 28 March 1988 Revised 10 August 1988 Accepted 11 August 1988 Received by publisher 16 August 1988
SUMMARY
Genes clockwise of oriC, the Escherichia coli replication origin (ort’C-mioC-asnC), show anticlockwise transcription. The intergenic region between mioC and asnC contains both a terminator and a consensus DnaA-protein-binding site (&PI box). We analysed termination in this region usinggaK expression to monitor for transcription. About 50% of the asnC transcripts were not terminated, and about 25% terminated at the asnC terminator. We found that the DnaA proteges box complex acts as a terminator of ~~sc~ption for about 25 y0 of the transcripts. Its efficiency could be increased by raising the level of DnaA protein, or it could be inactivated by deletion in the dnaA box or by thermal denatnration of the DnaA protein.
INTRODWCITON
The E. coli bidirectional origin of replication, OK, is located in an intergenic region between the mid and the gid genes (von Mey~b~g and Hansen, 1980). Adjacent to the mioC gene is the asnC gene (K0lling and Lother, 1985). Transcription of these Correspondenceto: Dr. W. Messer, Max-Planck-Institut Rir Molekulare Genetik, Ihnestrasse 73, D-1000 Berlin 33 (F.R.G.) Tel. (030)8307-266. Abbreviations: Ap, ampicillin; bla, j-lactamase; bp, base pair(s); dnuA box, site on DNA which binds the DnaA protein; D’IT, dithiothreitol; GalK, galactokinase; IPTG, isopropyl-@-r+ thiogalactoside; kb, kilobase or 1000 bp; nt, nucleotide(s); ori, origin of DNA replication; pos., position; P, promoter; r, terminator; t, time in minutes; wt, wild type.
genes is anticlockwise, the mioCand asnCtranscripts are synthesized toward oriC (reviewed in Messer, 1987). Upstream of the mioC promoter, in the intergenie region between mioC and asnC, is a consensus binding site for DnaA protein, the dnaA box, located close to the asnC t~ator (Hansen et al., 1982; Fnller et al., 1984). The binding of DnaA protein to the dnaA box leads to repression of transcription from the mioC promoter (Lother et al., 1985; Rokeach et al., 1986; Stuitje et al., 1986; Schauzu et al,, 1987; Lobner-Olesen et a)., 1987). DnaAregulated transcription originating at the mioC promoter increases the copy number of minicbromosomes (Stuitje et al., 1986; Lobner-Olesen et al., 1987). However, replication of minichromosomes initiates s~c~onously with the c~omosom~ origin, whether or not they contain the mioC region
0378-l 119/88/%03.50 0 1988 EIsevier Science Publishers B.V. (Biomedical Division)
348
(Hehnstetter and Leonard, 19X7), and the function of transcripts entering oriC, transcriptional activation or primer synthesis, is not known. Previous experiments on these transcripts did not discriminate between transcripts from mioC and those from the adjacent usnC gene. It was suggested that most of the usnC transcripts terminate at the as& terminator (Stuitje et al., 1986; Lsbner-Olesen et al., 1987). However, several observations suggest the existence of transcripts passing the asncterminator (Rokeach et al., 1986; KUlling et al., 1988; Gielow et al., 1988). The low rate of transcription distal to the a.snC terminator could be due to efficient repression of the asnC gene by the AsnC protein, encoded on high-copy-number vectors (K(llling and Lother, 1985; de Wind et al., 1985). To elucidate the elements involved in the regulation of termination of as& transcripts in the intergenie region between asnC and mioC, we cloned different DNA segments in front of the galK gene and measured the amount of transcripts passing different permutations of the intergenic region. We examined the efficiency of the asnC terminator and the effect of translation into the usnC terminator on the termination of transcription. The involvement of DnaA protein in the regulation of termination in the intergenic region was analysed by comparing transcript levels at varying concentrations of DnaA protein in the presence and absence of the dnu,4 box to which the DnaA protein binds cooperatively (Fuller et al., 1984).
MATERIALS AND METHODS
(a) Bacterial strains and plasmids Host strains for plasmids were Escherichia coli EC559 (ara-14, argE3, A(gpbproA)62, galK2, hk-4, lacY1, leuB6, mtl-1, rpsL31, supE44, thi-1, tsx-33, x$5), and EC558 (isogenic to EC559 but dnaA46) (Polaczek and Ciesla, 1984). The plasmid pHK27 (H. Kunze and W. M., unpublished), which carries the dnaA gene under the control of the tat promoter, was used for overexpression of DnaA protein. It is derived from plasmid pACYC184 by replacing the 0.69-kb HincII fragment by a 4.58 kb MstI fragment from pLSK5
(Schauzu et al., 1986), which contains lacIQ, the tat promoter fused to dnaA and rrnBtlt2. Plasmid pUTE13, used for the fusion of different DNA segments to the galactokinase gene, is derived from pKO1 (McKenney et al., 1981) by replacing the EcoRI-SmaI cloning region by the M 13mp 19 polylinker. Plasmid pLSK35-3 (Kalling and Lother, 1985) harbors the la& gene and the asnC gene from a BAL 31 generated end at nt position 1304 to MluI (nt pos. 777), fused to the tat promoter (all nt positions refer to the oriC sequence in Buhk and Messer, 1983). pLSK34-1 contains the tat promoter in front of the gafK gene (Lother et al., 1985). (b) Media For galactokinase and fi-lactamase assays cells were grown in M9 minimal medium supplemented with fructose (0.2%), casamino acids (0.5x), thiamine (10 pg/ml) and the necessary antibiotics Ap (50 pg/ml) or Cm (50 pg/ml), respectively. (c) In vitro construction of galK fusions Restriction enzymes were purchased from Boehringer Mannheim, Germany, mung bean nuclease from Pharmacia, and used as suggested by the manufacturer. Standard techniques for plasmid preparation and cloning were used (Maniatis et al., 1982). Plasmid pASN-1 harbors the asnC gene from nt position 1497 (HincII site) to nt position 782 (MluI site at nt pos. 777 treated with mung bean nuclease), integrated into the SmaI site of pUTE13. By insertion of the MnZI-HincII (nt pos. 660-1497) fragment into the SmaI site of pUTE13 we obtained pASN-2. pASN-3 (pASN-tat) contains the as& gene under the control of the tat promoter (Amann et al., 1983) in front of the gaZK gene (Fig. 1). It was obtained by cloning a 2024-bp ZVruI-PstI fragment with a truncated la& gene, the tat promoter and the asnC gene from pLSK35-3 into the SmaI-PstI sites ofpUTE13. Deletion of the PvuI site (nt pos. 937) in pASN-tat, by mung bean nuclease treatment of protruding PvuI ends and religation, yielded the plasmid pASN-4 (pASN-stop) with a deletion of 2 bp in the as&gene. The plasmid pASN-5 (pASN-box) contains a 6-bp deletion in the dnaA box (nt pos. 794-803). A MfuI-PvuI (nt pos. 777-937) fragment harboring this
deletion was obtained from plasmid pEM279 (Stuitje et al., 1986) and exchanged with the corresponding fragment in pASN-3. The plasmid with a deletion from the RsuI site at at position 839 to the RsaI site at nt position 917 in pASN-3 was named pASN-6 (pASN-pat for @al ierminator). This deletion included one-third of the WZC terminator. For easy reference pASN-3 through pASN-6 are designated pASN-tat, pASN-stop, pASN-pat and pASN-box, respectively, ~ou~out the rest of the paper. (d) ~aIact~~rn~
assays
The galactokinase assay procedures outlined by McKenney et al. (1981), were used with exception of the lysis of the cells. Overnight cultures in supplemented M9 medium were diluted 1~20~ into fresh medium and induced with IPTG (1 mM) at 30 ’ C in the case of plasmids which contained the tuc promoter in front of the asnC gene. When the culture reached an &e of 0.2-0.3 four samples were removed at 20 min intervals. One ml was mixed with 40 ~1 of lysis btier (500 mM Tris - HCI pH 8, 100 mM EDTA, 100 mM DTT) and 60 ~1 lysozyme (10 mgfml). This mixture was incubated for 30 min at 37 “C and further treated as described (McKenney et al., 1981).
galK units,,
= galK units x bla units-’
(see Table I).
Eis the extension of the coloured solution at the wave length of 490 nm. A,,, is the absorbance of the growing culture at 450 nm.
RESULTS
The intergenic region between the mioC and the asnC gene contains three elements, the mioC promoter, a dnaA box and the asnC terminator. We deleted different DNA segments to analyse the influence of these elements on ~~s~~ption. These difIerent mutant DNA segments were fused to the g& gene (McKermey et al., 1981) to measure the mount of transcripts passing the ~terge~c region. The plasmid pASN-1 harbors the asnC gene under the control of the asnC promoter and the complete intergenic region. pASN-2 contains in addition the mioC promoter. It has been suggested that asnC transcripts terminate efficiently at the asnC terminator (Stuitje et al., 1986; Lobner-Olesen et al., 1987). However, these results may be due to the autoregulation of the m&J gene (Kolling and Lother, 1985; De Wind et al., 1985). Therefore we replaced the autoregulated asnC promoter by the tat promoter (pASN-tat; Fig. 1).
(e) j&Lactamase assays (a) Term~tion To determine the relative copy number, we measured the activity of @lactamase encoded by the plasmids (S~gen~ 1968). 200 ~1 lysate (see above) were mixed with 2.5 ml phosphate buffer (100 mM, pH 6.5). After pr~cubation for 10 min at 3O”C, 0.5 ml penicillin G/K+, about 100~ units in H,O, were added and further incubated for 30 min at 30” C. This solution was rapidly mixed with 5.0 ml I, solution (1 vol. 0.32 M I, / 1.2 M KI solution and 19 ~01s. 0.5 M Na. acetate buffer, pH 4.0) and kept for 15 min at room temperature. Absorbance (A) was measured at 490 nm and converted into bla units : bla units = [s,,(without extract) - &@imp~e)l x 34.6’ x A450-1
The g&Z units were corrected for copy number according to :
between asnC and mid2
The amount of transcripts, which originate at the usnC promoter on pASN-1, was measured distal to the intergenic region, i.e., after the dnaA box and the asttC te~ator. No g&K activity was observed (Table I), corroborating earlier results (Stuitje et al., 1986; Laibner-Olesen et al., 1987). The plasmid pASN-2 with the mioC promoter served as a control and expressed 32 units of galactokinase. However when the asnC promoter in pASN- 1 was replaced by the tat promoter (pASN-tat), gaK expression of 76 units was observed. This is 54% of the gulK activity obtained with the control plasmid pLSK34- 1 (Table I). These results show that the reason for the low galK expression in pASN-1 is indeed the efficient autore~lation of the us& promoter and that the termination of u.snC transcription in the intergenic region is about 50% effective.
350
~A~N - stop -: pASN
pASN
Pi=
-pat
-
box
g&K
gaiK
+-----i
=
galK
galK
34-1 _.+_,
Ptac
517
a f..______..____..“-’
-___m__
Fig. 1, Map of the E. wli or&Yregion, with plasmid derivatives containing E. coii sequences from the intergenic region between arnC and mioC. Part A. Enlarged part of the intergenic region with the u.sK terminator and the &r& box. Thick line of the terminator indicates the translated part and ‘stop’ the position of the amber codon of the us& gene. Part B. Physical map of the atiC region. Filled bars represent the mioC, as& and a part of the us& gene. Filled arrows correspond to the positions of the respective promoters. The filled box adjacent to the r&C promoter indicates the dnaA box. Part C. Plasmids containing the subfragments of the iutergenic region msed to the g&K gene of pUTE13. The arrows indicate the direction of gawk expression. Plasmid pASN-1 contains the usxC gene with the complete intergenic region under the control of the natural u.rnC promoter. pASN-2 harbors in addition the mioC promoter and a part of the mioC gene. The exchange of the as& promoter with the rut promoter (arrowhead) leads to the following plasmids: pASN-tat corresponds to pASN-1 apart from the usnC promoter which is replaced by the fuc promoter. pASN-stop harbors a 2-bp deletion in the asnC gene which leads to a frame shift mutation resulting in a premature amber codon in front of the us&_?terminator. Stop codons are indicated by asterisks. pASN-pat contains a deletion of 78 bp between two RruI sites including one-third of the usnC terminator. pASN-box is a 6-bp deletion of pASN-tat Iocated in the dnuA box. pLSK34-I harbors the tat promoter in front of the g&K gene and serves as a control for maximum gaK expression.
(b)Effect of translatioo
OB
the efficiency of the asnC
terminator The stop codon of the usnC transcript is in the distal part of the possible stemloop structure which
forms the asnC terminator (Fig. 1). Since translation through a transcriptional terminator reduces its eff% ciency (Wright and Hayward, 1987), we prevented translation of ZkmC in the construct pASN-stop by a 2-bp deletion in the a.snC gene (nt pos. 936-937),
351 TABLE I Transcriptional activity passing the intergenic region between as& and mioC Plasmid”
GalK units b
Bla units b
GalK units,,,,=
pASN-1 pASN-2 pASN-tat pASN-stop pASN-pat pASN-box pLSK34-1
1 33 96 64 86 110 99
1.01 1.03 1.26 1.30 0.78 1.01 0.70
1 32 16 49 110 109 141
a The plasmids harboring the different fragments of the intergenic region are shown in Fig. 1. b Galactokinase units (galK units) and j-lactamase units (bla units) expressed as described in MATERIALS AND METHODS. c Galactokinase units corrected by the relative copy number, (B-lactamase units): galK units,,,, = galK units x bla units - r.
which caused a frame shift resulting in an amber codon in front of the DNA sequence coding for the stemloop structure. Plasmid pASN-stop expressed 35% less galactokinase than pASN-tat (Table I). (c) The role of the as& terminator To analyse the efficiency of the as& terminator we deleted a 78-bp RsuI fragment (nt pos. 917-839) of the as&gene, whereby one-third of the terminator was lost (pASN-pat; Fig. 1). By choice of these restriction sites an in-frame fusion was created, so that translation was allowed through the rest of the terminator up to the natural stop codon. Galactokinase expression from plasmid pASN-pat was 1.4-fold higher than expression from pASN-tat, but still about 25% lower than the expression from the control plasmid pLSK34- 1. These data suggest, that an additional factor is involved in the regulation of transcription in the intergenic region. One remarkable element in the intergenic region is the dnaA box. (d) The dnaA box affects transcription passing through the intergenic region Several DnaA protein monomers bind to the 9-bp consensus sequence (5’-TTITCCACA-3’), the dnaA box, located between TasnC and PmioC (Hansen et al., 1982; Fuller et al., 1984). This results in a repression of mioC transcription (Lother et al.,
1985; Stuitje et al., 1986; Rokeach et al., 1986; Schauzu et al., 1987; Larbner-Olesen et al., 1987). It is conceivable that DnaA protein binding to the dnaA box also terminates transcription from an upstream promoter (PusnC or Ptac), as has been described for a lac repressor/Zac operator complex (Deuschle et al., 1986). We analysed this effect on the DNA level by modifying the dnaA box and on the protein level using different amounts of active DnaA protein. In pASN-tat a fragment containing the natural dnaA box was replaced by a fragment with a dnaA box mutagenized in vitro in which 6 of 9 bp were deleted (Stuitje et al., 1986). The resulting plasmid, pASN-box, was analysed for guZK expression. The galK activity was about the same as with pASN-pat, i.e. about l.Cfold higher than the activity with pASN-tat (Table I). The deletion of each of the two structures, the asnC terminator and the dnaA box, respectively, resulted in about the same amount of increased transcription through the intergenic region, suggesting that both structures are about equally effective in termination in the presence of wildtype levels of DnaA protein. (e) Transcription termination at altered DnaA protein activity Transcription termination in the absence of DnaA protein was analysed by transforming the different plasmids into strain EC558, which is isogenic to EC559 except for the presence of the temperaturesensitive mutation dnaA46. The expression of galK was monitored after a shift from 30 ’ C to 42 ’ C (Fig. 2). The levels of expression at 30” C were identical to the galK activities in the dnaA + strain (compare t = 0 to Table I). These levels of gaZK activity did not change upon the temperature shift in plasmids without dnaA box (pLSK34-1, pASN-box). Upon inactivation of DnaA protein, pASN-tat gradually reached the expression level of pASN-box and pASN-pat the level of pLSK34-1, i.e. the activities of the comparable plasmids without dnaA box. In plasmid pASN-stop very little change in gulK activity was observed upon the shift to 42°C. Presumably, the DnaA protein mediated effect is masked in this case by the effective termination at the untranslated asnC terminator, TasnC. These results, obtained with inactivated DnaA protein, are corroborated by experiments using
352
DISCUSSION
~ 288
z5
z 2
158
58
icee
pASN-stop
I
p 150
1
28
---o-LI
Ti lee 50
o---
9
cn 288
48 60 t[ninl
t[ni Fig. 2. GalK synthesis upon inactivation of DnaA protein. EC558 (dnaA46) containing the different plasmids was grown in the presence of 1 mM IFTG at 30°C to A,,, = 0.2 and shifted to 42°C (t = 0). GaLK activity was monitored at ZO-minintervals. Galactokinase units were corrected for copy number using B-lactamase units.
elevated DnaA protein levels. EC558, containing the different pASN plasmids, was transformed with a second compatible plasmid, pHK27, which harbors the dnaA gene under the control of the tuc promoter which is itself controlled by the Ia@ gene on the same plasmid. Cultures were shifted from 30°C to 42 ’ C and at t = 0 both rut promoters (in front of the asnC gene and of the dnaA gene) were induced with 1 mM IPTG (Fig. 3). The final levels in g&K activity reached after 60 min induction with plasmids without dnaA box (pLSK34-1, pASN-box), were identical to the levels found in strains without pHK27. For plasmids with a dnaA box (pASN-tat, pASN-pat) guZK expression reached only about 60% of the activity found with wt amounts of DnaA protein. Expression on plasmid pASN-stop was close to background. These results show that two independent elements promote termination of transcription in the intergenic region: the asnC terminator and a complex between dnaA box and DnaA protein.
Analysis of as& transcription in high-copynumber promoter probe plasmids showed very low transcriptional activity distal to the asnC terminator. This was interpreted as efficient termination at TusnC (Stuitje et al., 1986; Lobner-Olesen et al., 1987). However, indirect observations (Rokeach et al., 1986) and Sl mapping experiments (Kblling et al., 1988; Gielow et al., 1988) demonstrate the existence of transcripts passing the usnC terminator. Here we showed that the low rate of transcription is due to the efficient repression of the asnC gene by AsnC protein (Kolling and Lother, 1985; De Wind et al., 1985). Replacement of the natural autoregulated asncpromoter by the tuc promoter resulted in 54% of the transcripts passing the intergenic region between asnC and mioC. This intergenic region harbors two independent structures which are effective in termination. One structure is the as& terminator, which resulted in about 25 % termination of transcription. The second structure is due to the interaction of DnaA protein with the dnaA box and also gave about 25% termination of transcription at wt levels of DnaA protein. The us& terminator harbors in the distal part the stop codon of the a.snCgene (Fig. 1). Translation into the stemloop was prevented by a frameshift mutation in the asnC gene. As suggested for p independent terminators (Wright and Hayward, 1987), interruption of translation caused 65 y0 of the transcripts to terminate, compared to 50% in plasmid pASNtat, in which the terminator is translated. DnaA protein bound to a dnaA box was functional as a transcription terminator. This was shown on the DNA level with plasmid pASN-box, in which the dnaA box was deleted. Compared to plasmid pASNtat harboring the dnaA box, transcription through the intergenic region increased l.Cfold. In order to analyse this regulatory structure on the protein level, we altered the amount of active DnaA protein. The rate of termination increased about two-fold at elevated DnaA protein levels (Fig. 3) in plasmids harboring the dnaA box (pASN-tat, pASN-pat) compared to the wt situation (Table I and Fig. 2, t = 0). In contrast, upon inactivation of DnaA protein, pASN-tat and pASN-pat showed increased galK activity, due to the decomposition of the dnaA box/DnaA protein complex. Plasmids pASN-box
353 I 288
_
2 15e5 188” g 59 e-d/, vI *#J
;
&---O-O
=
-
$$
lea
-
a
5o 8 2oo
E 158
150
I
?5 1tifJ
_ MN-stou
*;
I
(II 288 Pm-pat
pasn-tilt
/OF--O_
pASW-box
i
I
se_/-o---o-o e
28
I 48
68
ttnin1
t[ninl Fig. 3. GoK expression at elevated levels of DnaA protein. EC558 containing the DnaA-overproducing plasmid pHK27 and the diierent pASN plasmids was grown at 30°C. At t = 0 the strains were shifted to 42°C and IPTG (1 mM) was added, inducing the tuc promoter on the pASN plasmid and the tat promoter on the DnaA-overproducing plasmid pHK27. Galactokinase activity was measured in intervals of 20 min and corrected by the j?-lactamase units.
and pLSK34-1 lacking the dna-4 box were not affected. All these observations indicate that the dm4 box is involved in the regulation of transcribing RNA polymerase. The binding of DnaA protein to the dnaA box in the intergenic region caused the RNA polymerase to terminate. The observation that DnaA as a DNA-binding protein can block transcribing RNA polymerase and terminate trauscription is consistent with the observation of Deuschle et al. (1987). They demonstrated that the binding of lac repressor to a lac operator sequence which is distal and distant to a promoter results in termination of transcription in front of the luc repressorlluc operator complex. The asnC terminator and the dnuA box/DNA protein complex apparently terminate transcription independently of each other. Each of these elements could be separately inactivated without affecting the other structure, and the sum of the individual termination efficiencies corresponded approximately to
the total termination activity in this region. This is
corroborated by Sl mapping experiments, which have shown that at elevated DnaA protein concentrations termination increased close to the dnaA box, but termination at the as& terminator was unaffected (Gielow et al., 1988). In addition to these effects in close proximity to this dnaA box, DnaA protein has been found to change the termination pattern and the pausing of RNA polymerase within the mioC gene and oriC (Rokeach et al., 1986; 1987; Gielow et al., 1988). How and whether this effect influences initiation of replication in oriC remains to be elucidated. DnaA protein has been found to increase attenuation of the trp operon (Atlung and Hansen, 1983). However, there is no consensus DnaA binding site in the brp attenuator region. Therefore, the mechanism must be different from the one reported here. A number of genes harbor dnaA boxes at different positions. dm4 boxes close to promoters result in repression by DnaA protein as in case of the &a4 gene (Atlung et al., 1985; Braun et al., 1985; Kucherer et al., 1986) and the mioC gene (Lother et al., 1985; Rokeach et al., 1986; Stuitje et al., 1986; Lobner-Olesen et al., 1987). In the case of the dmA gene the dnaA box is located between promoter 1 and 2 (Hansen, F.G. et al., 1982). Both promoters are subject to autore~ation by DnaA protein (KUcherer et al., 1986). Therefore, we assume, that DnaA protein acts as a repressor for both promoters. However, we cannot exclude, that for promoter 1 the mechanism of autore8ulation is transcription termination. At dnuA boxes in transcribed but untranslated regions DnaA protein leads to transcription termination, as shown here. dnaA boxes are also located within the coding region of genes, e.g., within the M gene itself (Hansen et al., 1982). We may speculate that such genes are also regulated by binding of the DnaA protein to the internal dnaA boxes. They could terminate transcribing RNA polymerase and in this way prevent the expression of the gene products.
REFERENCES Amann, E, Brosius, J. and Ptashne, M.: Vectors bearing a hybrid frp-.&xc promoter useful for the regulated expression of cloned genes in E. [email protected] 25 (1983) 167-178.
354 Athmg, T., Clausen, E. and Hansen, F.G.: Autore8ulation of the dnaA gene of Esc?wkhia coli. Mol. Gen. Genet. 200 (1985) 442-450. Atlung, T. and Hansen, F.G.: Effect of dnaA and poB mutations on attenuation in the trp operon of Escherichia coli. J. Bacterlol. 156 (1983) 985-992. Braun, R.E., G’Day, K. and Wright, A.: Autor~ation of the DNA replication gene dnu.4 in E. coli. Cell 40 (1985) 159-I 69. Buhk, H.J. and Messer, W.: Replication origin region of EscRerichia coli: nucleotide sequence and functional units. Gene 24 (1983) 265-279. De Wind, N., de Jong, M., Meijer, M. and Stuitje, A.R.: Sitedirected mutagenesis of the Escherikhia coli chromosome near oriC: identification and characterization of us&, a regulatory element in Escherichia coli asparagine metabolism. Nucleic Acids Res. 13 (1985) 8797-8811. Deuschle, U., Gentz, R. and Bujard, H.: Zac repressor blocks transctibmg RNA polymerase and terminates transcription. Proc. Natl. Acad. Sci. USA 83 (1986) 4134-4137. Fuller, R.S., Funnell, B.E. and Komberg, A.: The &aA protein compfex with the E. coh’chromosomal origin (0%‘) and other sites. Ceil 38 (1984) 889-900. Gielow, A., Kttcherer, C., Ki%ing, R. and Messer, W.: Transcription in the region of the replication origin, oriC, of Escherichia cob. Termination of asnC transcripts. Mol. Gen. Genet. (1988) in press. Hansen, E.B., Hansen, F.G. and von Meyenburg, K.: The nucleotide sequence of the dnaA gene and the first part of the dnaN gene of Esche&hia coli K-12. Nucleic Acids Res. 10 (1982) 7373-7385. Hansen, F.G., Hansen, E.B. and Athmg, T.: The nucleotide sequence of the a%& gene promoter and of the adjacent rpmH gene, coding for the ribosomal protein L34, of F&e&h&s cd. EMBO J. 1 (1982) 1043-1048. Heimstetter, C.E. and Leonard, A.C.: Coordinate initiation of chromosome and minichromosome replication in Escherichia coli. J. Bacterial. 169 (1987) 3489-3494. Kolling, R., Gielow, A., Seufert, W., Klicherer, C. and Messer, W.: AsnC, a multifunctional regulator of genes located around the replication origin of Escherichia cob, oriC. Mol. Gen. Genet. 212 (1988) 99-104. Kolhng, R. and Lother, H.: As& an autogeneously regulated activator of asparagine synthetase A transcription in E. co& J. Bacterial. 164 11985) 310-315. Kticherer, C., Lother, H., KoBing,R., Schauzu, M.A. and Messer, W.: Regulation of ~~sc~ption of the chromosomal dnaA geneofEschetiiuc&. Mol. Gen. Genet. 205 (1986) 115-121. Lebner-Olesen, A., Atlung, T. and Rasmussen, K.V.: Stability and replication control of E. coli minichromosomes. J. Bacteriol. 169 (1987) 2835-2842.
Lother, H., Kolling, R., Kflcherer, C. and Schauzu, M.: Initiation of replication in Escherichia coli involves dnaA protein regulated transcription within the replication origin. EMBO J. 4 (1985) 555-560. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. McKenney, K., Shimatake, H., Court, D., Schmeissner, IJ., Brady, C. and Rosenberg, M.: A system to study promoter and terminator signals recognized by Escherichia coli RNA polymerase. In Chirikijan, J.G. and Papas, T.S. (Eds.) Gene Amplification and Analysis, Vol. 2: Analysis of Nucleic Acids by Enzymatic Methods. Elsevier/North Holland, Amsterdam, 1981, pp. 383-415. Messer, W.: Initiation of DNA replication in Escherichia cob. J. Bacterial. 169 (1987) 3395-3399. Polaczek, P. and Ciesla, 2.: Effect of altered efficiency of the RNA I and RNA II promoters on in vivo replication of ColEl-lie plasmids in Bscherichiu coli. Mol. Gen. Genet. 194 (1984) 227-231. Rokeach, L.A., Chiaramello, A., Junker, D.E., Cram, K., Nourani, A., Jannatipour, M. and Zyskind, J.W.: Effects of DnaA protein on replication and transcription events at the Escherichia coli origin of replication, oriC. In McMacken, R. and Kelly, T.J. (Eds.), DNA Replication and Recombination. Liss, New York, 1986, pp. 415-427. Rokeach, L.A., Kassavetis, G.A. and Zyskind, J.W.: RNA polymerase pauses in vitro within the Escherichia coli origin of replication at the same sites where termination occurs in vivo. J. Biol. Chem. 262 (1987) 7264-7272. Sargent, M.G.: Rapid fixed-time assay for penicillinase. J. Bacterial. 95 (1968) 1493-1494. Schauzu, M.A., Kticherer, C., Kiflling, R., Messer, W. and Lother, H.: Transcripts within the replication origin, orX’, of Escherichia cob. Nucleic Acids Res. 15 ( 1987) 2479-2497. Stuitje, A.R., De Wind, N., Van der Spek, J.C., Pors, T.H. and Meijer, M.: Dissection of promoter sequences involved in transcriptional activation of the Escherichia coli replication origin. Nucleic Acids Res. 14 (1986) 2333-2344. von Meyenburg, K. and Hansen, F.G.: The origin of replication, oriC, of the E. coli chromosome: genes near oriC and construction of oriC deletion mutations. ICN-UCLA Symp. Mol. Cell. Biol. 19 (1980) 137-159. Wright, J. and Hayward, R.: Tr~s~~ption~ termination at a fully rho-~dependent site in E. co& is prevented by uninterrupted translation of the nascent RNA. EMBO J. 6 (1987) 1115-1119. Communicated by M. Salas.
Gene, 73 (1988) 347-3.54
Elsevier GEN 02763
Termination of the EsclrerMGa coii us& transwipt. The DnaA proteim/dmzA box complex blocks transcribiig RNA plymerase (Initiation of replication; oriC; mioC; galhl-expression plasmid; autore~ation;
recombinant DNA)
Christoph Schaefer and Walter Messer Max-Planck-Institut fiir Molekulare Gene&, Berlin (F.R.G.) Received 28 March 1988 Revised 10 August 1988 Accepted 11 August 1988 Received by publisher 16 August 1988
SUMMARY
Genes clockwise of oriC, the Escherichia coli replication origin (ort’C-mioC-asnC), show anticlockwise transcription. The intergenic region between mioC and asnC contains both a terminator and a consensus DnaA-protein-binding site (&PI box). We analysed termination in this region usinggaK expression to monitor for transcription. About 50% of the asnC transcripts were not terminated, and about 25% terminated at the asnC terminator. We found that the DnaA proteges box complex acts as a terminator of ~~sc~ption for about 25 y0 of the transcripts. Its efficiency could be increased by raising the level of DnaA protein, or it could be inactivated by deletion in the dnaA box or by thermal denatnration of the DnaA protein.
INTRODWCITON
The E. coli bidirectional origin of replication, OK, is located in an intergenic region between the mid and the gid genes (von Mey~b~g and Hansen, 1980). Adjacent to the mioC gene is the asnC gene (K0lling and Lother, 1985). Transcription of these Correspondenceto: Dr. W. Messer, Max-Planck-Institut Rir Molekulare Genetik, Ihnestrasse 73, D-1000 Berlin 33 (F.R.G.) Tel. (030)8307-266. Abbreviations: Ap, ampicillin; bla, j-lactamase; bp, base pair(s); dnuA box, site on DNA which binds the DnaA protein; D’IT, dithiothreitol; GalK, galactokinase; IPTG, isopropyl-@-r+ thiogalactoside; kb, kilobase or 1000 bp; nt, nucleotide(s); ori, origin of DNA replication; pos., position; P, promoter; r, terminator; t, time in minutes; wt, wild type.
genes is anticlockwise, the mioCand asnCtranscripts are synthesized toward oriC (reviewed in Messer, 1987). Upstream of the mioC promoter, in the intergenie region between mioC and asnC, is a consensus binding site for DnaA protein, the dnaA box, located close to the asnC t~ator (Hansen et al., 1982; Fnller et al., 1984). The binding of DnaA protein to the dnaA box leads to repression of transcription from the mioC promoter (Lother et al., 1985; Rokeach et al., 1986; Stuitje et al., 1986; Schauzu et al,, 1987; Lobner-Olesen et a)., 1987). DnaAregulated transcription originating at the mioC promoter increases the copy number of minicbromosomes (Stuitje et al., 1986; Lobner-Olesen et al., 1987). However, replication of minichromosomes initiates s~c~onously with the c~omosom~ origin, whether or not they contain the mioC region
0378-l 119/88/%03.50 0 1988 EIsevier Science Publishers B.V. (Biomedical Division)
348
(Hehnstetter and Leonard, 19X7), and the function of transcripts entering oriC, transcriptional activation or primer synthesis, is not known. Previous experiments on these transcripts did not discriminate between transcripts from mioC and those from the adjacent usnC gene. It was suggested that most of the usnC transcripts terminate at the as& terminator (Stuitje et al., 1986; Lsbner-Olesen et al., 1987). However, several observations suggest the existence of transcripts passing the asncterminator (Rokeach et al., 1986; KUlling et al., 1988; Gielow et al., 1988). The low rate of transcription distal to the a.snC terminator could be due to efficient repression of the asnC gene by the AsnC protein, encoded on high-copy-number vectors (K(llling and Lother, 1985; de Wind et al., 1985). To elucidate the elements involved in the regulation of termination of as& transcripts in the intergenie region between asnC and mioC, we cloned different DNA segments in front of the galK gene and measured the amount of transcripts passing different permutations of the intergenic region. We examined the efficiency of the asnC terminator and the effect of translation into the usnC terminator on the termination of transcription. The involvement of DnaA protein in the regulation of termination in the intergenic region was analysed by comparing transcript levels at varying concentrations of DnaA protein in the presence and absence of the dnu,4 box to which the DnaA protein binds cooperatively (Fuller et al., 1984).
MATERIALS AND METHODS
(a) Bacterial strains and plasmids Host strains for plasmids were Escherichia coli EC559 (ara-14, argE3, A(gpbproA)62, galK2, hk-4, lacY1, leuB6, mtl-1, rpsL31, supE44, thi-1, tsx-33, x$5), and EC558 (isogenic to EC559 but dnaA46) (Polaczek and Ciesla, 1984). The plasmid pHK27 (H. Kunze and W. M., unpublished), which carries the dnaA gene under the control of the tat promoter, was used for overexpression of DnaA protein. It is derived from plasmid pACYC184 by replacing the 0.69-kb HincII fragment by a 4.58 kb MstI fragment from pLSK5
(Schauzu et al., 1986), which contains lacIQ, the tat promoter fused to dnaA and rrnBtlt2. Plasmid pUTE13, used for the fusion of different DNA segments to the galactokinase gene, is derived from pKO1 (McKenney et al., 1981) by replacing the EcoRI-SmaI cloning region by the M 13mp 19 polylinker. Plasmid pLSK35-3 (Kalling and Lother, 1985) harbors the la& gene and the asnC gene from a BAL 31 generated end at nt position 1304 to MluI (nt pos. 777), fused to the tat promoter (all nt positions refer to the oriC sequence in Buhk and Messer, 1983). pLSK34-1 contains the tat promoter in front of the gafK gene (Lother et al., 1985). (b) Media For galactokinase and fi-lactamase assays cells were grown in M9 minimal medium supplemented with fructose (0.2%), casamino acids (0.5x), thiamine (10 pg/ml) and the necessary antibiotics Ap (50 pg/ml) or Cm (50 pg/ml), respectively. (c) In vitro construction of galK fusions Restriction enzymes were purchased from Boehringer Mannheim, Germany, mung bean nuclease from Pharmacia, and used as suggested by the manufacturer. Standard techniques for plasmid preparation and cloning were used (Maniatis et al., 1982). Plasmid pASN-1 harbors the asnC gene from nt position 1497 (HincII site) to nt position 782 (MluI site at nt pos. 777 treated with mung bean nuclease), integrated into the SmaI site of pUTE13. By insertion of the MnZI-HincII (nt pos. 660-1497) fragment into the SmaI site of pUTE13 we obtained pASN-2. pASN-3 (pASN-tat) contains the as& gene under the control of the tat promoter (Amann et al., 1983) in front of the gaZK gene (Fig. 1). It was obtained by cloning a 2024-bp ZVruI-PstI fragment with a truncated la& gene, the tat promoter and the asnC gene from pLSK35-3 into the SmaI-PstI sites ofpUTE13. Deletion of the PvuI site (nt pos. 937) in pASN-tat, by mung bean nuclease treatment of protruding PvuI ends and religation, yielded the plasmid pASN-4 (pASN-stop) with a deletion of 2 bp in the as&gene. The plasmid pASN-5 (pASN-box) contains a 6-bp deletion in the dnaA box (nt pos. 794-803). A MfuI-PvuI (nt pos. 777-937) fragment harboring this
deletion was obtained from plasmid pEM279 (Stuitje et al., 1986) and exchanged with the corresponding fragment in pASN-3. The plasmid with a deletion from the RsuI site at at position 839 to the RsaI site at nt position 917 in pASN-3 was named pASN-6 (pASN-pat for @al ierminator). This deletion included one-third of the WZC terminator. For easy reference pASN-3 through pASN-6 are designated pASN-tat, pASN-stop, pASN-pat and pASN-box, respectively, ~ou~out the rest of the paper. (d) ~aIact~~rn~
assays
The galactokinase assay procedures outlined by McKenney et al. (1981), were used with exception of the lysis of the cells. Overnight cultures in supplemented M9 medium were diluted 1~20~ into fresh medium and induced with IPTG (1 mM) at 30 ’ C in the case of plasmids which contained the tuc promoter in front of the asnC gene. When the culture reached an &e of 0.2-0.3 four samples were removed at 20 min intervals. One ml was mixed with 40 ~1 of lysis btier (500 mM Tris - HCI pH 8, 100 mM EDTA, 100 mM DTT) and 60 ~1 lysozyme (10 mgfml). This mixture was incubated for 30 min at 37 “C and further treated as described (McKenney et al., 1981).
galK units,,
= galK units x bla units-’
(see Table I).
Eis the extension of the coloured solution at the wave length of 490 nm. A,,, is the absorbance of the growing culture at 450 nm.
RESULTS
The intergenic region between the mioC and the asnC gene contains three elements, the mioC promoter, a dnaA box and the asnC terminator. We deleted different DNA segments to analyse the influence of these elements on ~~s~~ption. These difIerent mutant DNA segments were fused to the g& gene (McKermey et al., 1981) to measure the mount of transcripts passing the ~terge~c region. The plasmid pASN-1 harbors the asnC gene under the control of the asnC promoter and the complete intergenic region. pASN-2 contains in addition the mioC promoter. It has been suggested that asnC transcripts terminate efficiently at the asnC terminator (Stuitje et al., 1986; Lobner-Olesen et al., 1987). However, these results may be due to the autoregulation of the m&J gene (Kolling and Lother, 1985; De Wind et al., 1985). Therefore we replaced the autoregulated asnC promoter by the tat promoter (pASN-tat; Fig. 1).
(e) j&Lactamase assays (a) Term~tion To determine the relative copy number, we measured the activity of @lactamase encoded by the plasmids (S~gen~ 1968). 200 ~1 lysate (see above) were mixed with 2.5 ml phosphate buffer (100 mM, pH 6.5). After pr~cubation for 10 min at 3O”C, 0.5 ml penicillin G/K+, about 100~ units in H,O, were added and further incubated for 30 min at 30” C. This solution was rapidly mixed with 5.0 ml I, solution (1 vol. 0.32 M I, / 1.2 M KI solution and 19 ~01s. 0.5 M Na. acetate buffer, pH 4.0) and kept for 15 min at room temperature. Absorbance (A) was measured at 490 nm and converted into bla units : bla units = [s,,(without extract) - &@imp~e)l x 34.6’ x A450-1
The g&Z units were corrected for copy number according to :
between asnC and mid2
The amount of transcripts, which originate at the usnC promoter on pASN-1, was measured distal to the intergenic region, i.e., after the dnaA box and the asttC te~ator. No g&K activity was observed (Table I), corroborating earlier results (Stuitje et al., 1986; Laibner-Olesen et al., 1987). The plasmid pASN-2 with the mioC promoter served as a control and expressed 32 units of galactokinase. However when the asnC promoter in pASN- 1 was replaced by the tat promoter (pASN-tat), gaK expression of 76 units was observed. This is 54% of the gulK activity obtained with the control plasmid pLSK34- 1 (Table I). These results show that the reason for the low galK expression in pASN-1 is indeed the efficient autore~lation of the us& promoter and that the termination of u.snC transcription in the intergenic region is about 50% effective.
350
~A~N - stop -: pASN
pASN
Pi=
-pat
-
box
g&K
gaiK
+-----i
=
galK
galK
34-1 _.+_,
Ptac
517
a f..______..____..“-’
-___m__
Fig. 1, Map of the E. wli or&Yregion, with plasmid derivatives containing E. coii sequences from the intergenic region between arnC and mioC. Part A. Enlarged part of the intergenic region with the u.sK terminator and the &r& box. Thick line of the terminator indicates the translated part and ‘stop’ the position of the amber codon of the us& gene. Part B. Physical map of the atiC region. Filled bars represent the mioC, as& and a part of the us& gene. Filled arrows correspond to the positions of the respective promoters. The filled box adjacent to the r&C promoter indicates the dnaA box. Part C. Plasmids containing the subfragments of the iutergenic region msed to the g&K gene of pUTE13. The arrows indicate the direction of gawk expression. Plasmid pASN-1 contains the usxC gene with the complete intergenic region under the control of the natural u.rnC promoter. pASN-2 harbors in addition the mioC promoter and a part of the mioC gene. The exchange of the as& promoter with the rut promoter (arrowhead) leads to the following plasmids: pASN-tat corresponds to pASN-1 apart from the usnC promoter which is replaced by the fuc promoter. pASN-stop harbors a 2-bp deletion in the asnC gene which leads to a frame shift mutation resulting in a premature amber codon in front of the us&_?terminator. Stop codons are indicated by asterisks. pASN-pat contains a deletion of 78 bp between two RruI sites including one-third of the usnC terminator. pASN-box is a 6-bp deletion of pASN-tat Iocated in the dnuA box. pLSK34-I harbors the tat promoter in front of the g&K gene and serves as a control for maximum gaK expression.
(b)Effect of translatioo
OB
the efficiency of the asnC
terminator The stop codon of the usnC transcript is in the distal part of the possible stemloop structure which
forms the asnC terminator (Fig. 1). Since translation through a transcriptional terminator reduces its eff% ciency (Wright and Hayward, 1987), we prevented translation of ZkmC in the construct pASN-stop by a 2-bp deletion in the a.snC gene (nt pos. 936-937),
351 TABLE I Transcriptional activity passing the intergenic region between as& and mioC Plasmid”
GalK units b
Bla units b
GalK units,,,,=
pASN-1 pASN-2 pASN-tat pASN-stop pASN-pat pASN-box pLSK34-1
1 33 96 64 86 110 99
1.01 1.03 1.26 1.30 0.78 1.01 0.70
1 32 16 49 110 109 141
a The plasmids harboring the different fragments of the intergenic region are shown in Fig. 1. b Galactokinase units (galK units) and j-lactamase units (bla units) expressed as described in MATERIALS AND METHODS. c Galactokinase units corrected by the relative copy number, (B-lactamase units): galK units,,,, = galK units x bla units - r.
which caused a frame shift resulting in an amber codon in front of the DNA sequence coding for the stemloop structure. Plasmid pASN-stop expressed 35% less galactokinase than pASN-tat (Table I). (c) The role of the as& terminator To analyse the efficiency of the as& terminator we deleted a 78-bp RsuI fragment (nt pos. 917-839) of the as&gene, whereby one-third of the terminator was lost (pASN-pat; Fig. 1). By choice of these restriction sites an in-frame fusion was created, so that translation was allowed through the rest of the terminator up to the natural stop codon. Galactokinase expression from plasmid pASN-pat was 1.4-fold higher than expression from pASN-tat, but still about 25% lower than the expression from the control plasmid pLSK34- 1. These data suggest, that an additional factor is involved in the regulation of transcription in the intergenic region. One remarkable element in the intergenic region is the dnaA box. (d) The dnaA box affects transcription passing through the intergenic region Several DnaA protein monomers bind to the 9-bp consensus sequence (5’-TTITCCACA-3’), the dnaA box, located between TasnC and PmioC (Hansen et al., 1982; Fuller et al., 1984). This results in a repression of mioC transcription (Lother et al.,
1985; Stuitje et al., 1986; Rokeach et al., 1986; Schauzu et al., 1987; Larbner-Olesen et al., 1987). It is conceivable that DnaA protein binding to the dnaA box also terminates transcription from an upstream promoter (PusnC or Ptac), as has been described for a lac repressor/Zac operator complex (Deuschle et al., 1986). We analysed this effect on the DNA level by modifying the dnaA box and on the protein level using different amounts of active DnaA protein. In pASN-tat a fragment containing the natural dnaA box was replaced by a fragment with a dnaA box mutagenized in vitro in which 6 of 9 bp were deleted (Stuitje et al., 1986). The resulting plasmid, pASN-box, was analysed for guZK expression. The galK activity was about the same as with pASN-pat, i.e. about l.Cfold higher than the activity with pASN-tat (Table I). The deletion of each of the two structures, the asnC terminator and the dnaA box, respectively, resulted in about the same amount of increased transcription through the intergenic region, suggesting that both structures are about equally effective in termination in the presence of wildtype levels of DnaA protein. (e) Transcription termination at altered DnaA protein activity Transcription termination in the absence of DnaA protein was analysed by transforming the different plasmids into strain EC558, which is isogenic to EC559 except for the presence of the temperaturesensitive mutation dnaA46. The expression of galK was monitored after a shift from 30 ’ C to 42 ’ C (Fig. 2). The levels of expression at 30” C were identical to the galK activities in the dnaA + strain (compare t = 0 to Table I). These levels of gaZK activity did not change upon the temperature shift in plasmids without dnaA box (pLSK34-1, pASN-box). Upon inactivation of DnaA protein, pASN-tat gradually reached the expression level of pASN-box and pASN-pat the level of pLSK34-1, i.e. the activities of the comparable plasmids without dnaA box. In plasmid pASN-stop very little change in gulK activity was observed upon the shift to 42°C. Presumably, the DnaA protein mediated effect is masked in this case by the effective termination at the untranslated asnC terminator, TasnC. These results, obtained with inactivated DnaA protein, are corroborated by experiments using
352
DISCUSSION
~ 288
z5
z 2
158
58
icee
pASN-stop
I
p 150
1
28
---o-LI
Ti lee 50
o---
9
cn 288
48 60 t[ninl
t[ni Fig. 2. GalK synthesis upon inactivation of DnaA protein. EC558 (dnaA46) containing the different plasmids was grown in the presence of 1 mM IFTG at 30°C to A,,, = 0.2 and shifted to 42°C (t = 0). GaLK activity was monitored at ZO-minintervals. Galactokinase units were corrected for copy number using B-lactamase units.
elevated DnaA protein levels. EC558, containing the different pASN plasmids, was transformed with a second compatible plasmid, pHK27, which harbors the dnaA gene under the control of the tuc promoter which is itself controlled by the Ia@ gene on the same plasmid. Cultures were shifted from 30°C to 42 ’ C and at t = 0 both rut promoters (in front of the asnC gene and of the dnaA gene) were induced with 1 mM IPTG (Fig. 3). The final levels in g&K activity reached after 60 min induction with plasmids without dnaA box (pLSK34-1, pASN-box), were identical to the levels found in strains without pHK27. For plasmids with a dnaA box (pASN-tat, pASN-pat) guZK expression reached only about 60% of the activity found with wt amounts of DnaA protein. Expression on plasmid pASN-stop was close to background. These results show that two independent elements promote termination of transcription in the intergenic region: the asnC terminator and a complex between dnaA box and DnaA protein.
Analysis of as& transcription in high-copynumber promoter probe plasmids showed very low transcriptional activity distal to the asnC terminator. This was interpreted as efficient termination at TusnC (Stuitje et al., 1986; Lobner-Olesen et al., 1987). However, indirect observations (Rokeach et al., 1986) and Sl mapping experiments (Kblling et al., 1988; Gielow et al., 1988) demonstrate the existence of transcripts passing the usnC terminator. Here we showed that the low rate of transcription is due to the efficient repression of the asnC gene by AsnC protein (Kolling and Lother, 1985; De Wind et al., 1985). Replacement of the natural autoregulated asncpromoter by the tuc promoter resulted in 54% of the transcripts passing the intergenic region between asnC and mioC. This intergenic region harbors two independent structures which are effective in termination. One structure is the as& terminator, which resulted in about 25 % termination of transcription. The second structure is due to the interaction of DnaA protein with the dnaA box and also gave about 25% termination of transcription at wt levels of DnaA protein. The us& terminator harbors in the distal part the stop codon of the a.snCgene (Fig. 1). Translation into the stemloop was prevented by a frameshift mutation in the asnC gene. As suggested for p independent terminators (Wright and Hayward, 1987), interruption of translation caused 65 y0 of the transcripts to terminate, compared to 50% in plasmid pASNtat, in which the terminator is translated. DnaA protein bound to a dnaA box was functional as a transcription terminator. This was shown on the DNA level with plasmid pASN-box, in which the dnaA box was deleted. Compared to plasmid pASNtat harboring the dnaA box, transcription through the intergenic region increased l.Cfold. In order to analyse this regulatory structure on the protein level, we altered the amount of active DnaA protein. The rate of termination increased about two-fold at elevated DnaA protein levels (Fig. 3) in plasmids harboring the dnaA box (pASN-tat, pASN-pat) compared to the wt situation (Table I and Fig. 2, t = 0). In contrast, upon inactivation of DnaA protein, pASN-tat and pASN-pat showed increased galK activity, due to the decomposition of the dnaA box/DnaA protein complex. Plasmids pASN-box
353 I 288
_
2 15e5 188” g 59 e-d/, vI *#J
;
&---O-O
=
-
$$
lea
-
a
5o 8 2oo
E 158
150
I
?5 1tifJ
_ MN-stou
*;
I
(II 288 Pm-pat
pasn-tilt
/OF--O_
pASW-box
i
I
se_/-o---o-o e
28
I 48
68
ttnin1
t[ninl Fig. 3. GoK expression at elevated levels of DnaA protein. EC558 containing the DnaA-overproducing plasmid pHK27 and the diierent pASN plasmids was grown at 30°C. At t = 0 the strains were shifted to 42°C and IPTG (1 mM) was added, inducing the tuc promoter on the pASN plasmid and the tat promoter on the DnaA-overproducing plasmid pHK27. Galactokinase activity was measured in intervals of 20 min and corrected by the j?-lactamase units.
and pLSK34-1 lacking the dna-4 box were not affected. All these observations indicate that the dm4 box is involved in the regulation of transcribing RNA polymerase. The binding of DnaA protein to the dnaA box in the intergenic region caused the RNA polymerase to terminate. The observation that DnaA as a DNA-binding protein can block transcribing RNA polymerase and terminate trauscription is consistent with the observation of Deuschle et al. (1987). They demonstrated that the binding of lac repressor to a lac operator sequence which is distal and distant to a promoter results in termination of transcription in front of the luc repressorlluc operator complex. The asnC terminator and the dnuA box/DNA protein complex apparently terminate transcription independently of each other. Each of these elements could be separately inactivated without affecting the other structure, and the sum of the individual termination efficiencies corresponded approximately to
the total termination activity in this region. This is
corroborated by Sl mapping experiments, which have shown that at elevated DnaA protein concentrations termination increased close to the dnaA box, but termination at the as& terminator was unaffected (Gielow et al., 1988). In addition to these effects in close proximity to this dnaA box, DnaA protein has been found to change the termination pattern and the pausing of RNA polymerase within the mioC gene and oriC (Rokeach et al., 1986; 1987; Gielow et al., 1988). How and whether this effect influences initiation of replication in oriC remains to be elucidated. DnaA protein has been found to increase attenuation of the trp operon (Atlung and Hansen, 1983). However, there is no consensus DnaA binding site in the brp attenuator region. Therefore, the mechanism must be different from the one reported here. A number of genes harbor dnaA boxes at different positions. dm4 boxes close to promoters result in repression by DnaA protein as in case of the &a4 gene (Atlung et al., 1985; Braun et al., 1985; Kucherer et al., 1986) and the mioC gene (Lother et al., 1985; Rokeach et al., 1986; Stuitje et al., 1986; Lobner-Olesen et al., 1987). In the case of the dmA gene the dnaA box is located between promoter 1 and 2 (Hansen, F.G. et al., 1982). Both promoters are subject to autore~ation by DnaA protein (KUcherer et al., 1986). Therefore, we assume, that DnaA protein acts as a repressor for both promoters. However, we cannot exclude, that for promoter 1 the mechanism of autore8ulation is transcription termination. At dnuA boxes in transcribed but untranslated regions DnaA protein leads to transcription termination, as shown here. dnaA boxes are also located within the coding region of genes, e.g., within the M gene itself (Hansen et al., 1982). We may speculate that such genes are also regulated by binding of the DnaA protein to the internal dnaA boxes. They could terminate transcribing RNA polymerase and in this way prevent the expression of the gene products.
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