JOURNAL OF BACrERIOLOGY, Nov. 1992, p. 6862-6871
Vol. 174, No. 21
0021-9193/92/216862-10$02.00/0 Copyright © 1992, American Society for Microbiology
Sequence Elements in the Escherichia coli araFGH Promoter WILLIAM HENDRICKSON,* CLAIRE FLAHERTY,t AND LISA MOLZ Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, P. O. Box 6998, Chicago, Illinois 60680 Received 1 May 1992/Accepted 27 August 1992
The Eschcrichia coli araFGH operon codes for proteins involved in the L-arabinose high-affinity transport system. Transcriptional regulation of the operon was studied by creating point mutations and deletions in the control region cloned into a GalK expression vector. The transcription start site was confirmed by RNA sequencing of transcripts. The sequences essential for polymerase function were localized by deletions and point mutations. Surprisingly, only a weak -10 consensus sequence, and no -35 sequence is required. Mutation of a guanosine at position -12 greatly reduced promoter activity, which suggests important polymerase interactions with DNA between the usual -10 and -35 positions. A double mutation toward the consensus in the -10 region was required to create a promoter capable of significant AraC-independent transcription. These results show that the araFGH promoter structure is similar to that of the galPI promoter and is substantially different from that of the araBAD promoter. The effects of 11 mutations within the DNA region thought to bind the cyclic AMP receptor protein correlate well with the CRP consensus binding sequence and confirm that this region is responsible for cyclic AMP regulation. Deletion of the AraC binding site nearest the promoter, araFGl, eliminates arabinose regulation, whereas deletion of the upstream AraC binding site, araFG2, has only a slight effect on promoter activity.
associated and have regions of strong sequence similarity with other proteins involved in sugar transport (31, 58). Both transport operons, as well as the operon, araBAD, for the catabolic enzymes are regulated at the initiation of transcription by the intracellular levels of cyclic AMP (cAMP) and L-arabinose (15, 22, 37, 66). The cAMP receptor protein (CRP) is responsible for catabolic regulation in response to cAMP, and the AraC protein responds to L-arabinose. Both of these activators bind adjacent sites of the promoters just upstream from the RNA polymerase binding region. At the araFGH promoter, however, the orientation of the sites is reversed as compared with those of the other ara promoters; the CRP site is adjacent to that for RNA polymerase, and AraC binds upstream of CRP (Fig. 1) (29), whereas at araBAD AraC binds adjacent to polymerase and CRP binds upstream (48). These promoter structures help explain some of the differences between the ara promoters with regard to regulation. araFGH is highly sensitive to catabolite repression and is not capable of significant transcription in the absence of CRP either in vivo or in vitro (29, 37). On the other hand, araBAD is only moderately regulated by CRP and can be substantially induced by arabinose-AraC alone. Transcription of the araBAD operon is repressed by the action of AraC protein in a looped DNA complex with a single molecule of AraC bound to an upstream site, araO2, and to the inducer site, araI (33, 40, 43). CRP appears to activate transcription directly and also indirectly by breaking the repression loop (23, 24, 39, 41, 61). It has been suggested that araFGH is also repressed by AraC protein in the absence of arabinose (37). The araBAD promoter has recognizable -10 and -35 hexamers. Mutations that decrease transcription, or increase transcription in the absence of activators, have been found at both sites (10, 20, 23). In the study reported here, we performed the first genetic analysis of the araFGH promoter. The promoter region was fused to the galactokinase gene to isolate mutants with decreased or increased promoter function. The mutants were characterized with respect to the induced and basal levels of expression. These
The RNA polymerase binding sites of many promoters have been studied in detail. A consensus sequence for recognition by RNA polymerase c7o includes two conserved hexanucleotide sequences located 10 and 35 bp upstream from the transcription start site (25, 26, 46, 67). Additional sequences between and upstream of these elements are weakly conserved but can significantly affect promoter strength (2, 50). A model of polymerase recognition based on genetic experiments showing interactions of different amino acid motifs within the cr subunit with specific base pairs of the -10 and -35 regions has been developed (18, 60, 65). Our present knowledge of promoter structure is based primarily on the analysis of strong promoters that do not require accessory proteins. Promoters requiring activators frequently have a -35 region with poor similarity to the consensus (25). It is not clear, however, whether these promoters are simply characterized by a poor polymerase recognition site or whether they contain unique recognition elements. We are studying the Escherichia coli promoters that regulate genes involved in arabinose metabolism to understand structure-function relationships of promoters that require multiple activators for transcription. T`wo distinct systems are capable of active transport of the sugar L-arabinose in E. coli. A low-affinity, high-capacity symporter system is the product of the araE operon (6, 36, 62). At present only one protein is known to be encoded by this operon. A high-affinity system is encoded by the araFGH operon located at 41.9 min (2,000 bp) on the E. cdli map (9, 27, 37, 58). The product of the araF gene, L-arabinose binding protein, is located in the periplasm and is required for high-affinity transport (30, 55), and two additional genes of unknown function, araG and araH, have been identified. Both araG and araH products are membrane *
Corresponding author.
t Present address: Department of Anatomy and Neuroscience, Pennsylvania State University College of Medicine, Hershey, PA 17033. 6862
araFGH PROMOTER
VOL. 174, 1992 -155
_ AraC
-80
41
araF
AraC CRP
RNA pol.
araFGl araFG2 FIG. 1. Control region of the araFGH promoter. Binding sites for AraC protein, CRP, and RNA polymerase are shown. The distance from the transcription start site (arrow) to the center of each activator protein is given above the diagram. In plasmid pWH54, the 505-bp DNA fragment containing the control region is fused to galK via a HindIII linker at position +90.
mutants allowed
us to clearly identify the protein binding sites and show that the RNA polymerase binding site is much different than that suggested for the araBAD promoter; only minimal polymerase recognition elements are present, and an important recognition element lies adjacent to the -10 region.
MATERIALS AND METHODS General procedures. pWH54 contains the araFGH proon a 500-bp HindIII-EcoRI fragment (Fig. 1) (29). All plasmids were introduced by transformation into strain WH75 (F- leu Alac-74 galK thi araB, Strr), which is araC+ and araBAD. WH75 was constructed by P1 transduction of the polar araB mutation of SH313 into E. coli K-12 strain SH2. Gel electrophoresis band shift assays were performed as described previously (28). A 235-bp restriction fragment of pWH54 was end labeled with 32P and used in the gel-shift assay. All general DNA manipulations, restriction digestions, ligations, and DNA transformations were as described previously (53, 56). Galactokinase assays. Cultures (20 ml) were grown in M9 medium (53) plus 4 p,g of thiamine per ml, 0.2% Casamino Acids, and 20 pg of ampicillin per ml at 37°C to an A650 of 0.6. Galactokinase assays were performed on toluenetreated cells by the method of McKenney et al. (44). In this assay the production of [14C]galactose phosphate is assayed by its retention on DEAE filters (Whatman DE81). Units are millimoles of galactose phosphate produced per minute per milliliter of culture (A650, 1). Determinations were made in triplicate for each culture, with at least two separate cultures utilized for each strain. Cells were induced by adding 0.2% arabinose to the cultures for at least four generations. Parallel ampicillinase measurements by the nitroceffin assay (63) were performed on a portion of the toluenetreated cells of each culture to insure that the plasmid copy number remained constant. The reaction of ampicillin from the permeabilized cells with nitroceffin was measured by the increase inA492. The copy number of the pKO1 system used for these studies has been reported to vary when actively transcribed genes are present (1). We carefully tested the constructs and found that transcription from the araFGH promoter is not sufficient to alter the copy number. All of the mutants had the same copy number (±10%) as the wild type within the error of the assay. Occasional cultures with significant variations in copy number were discarded. Since multiple cultures were done for each mutant, the error due to random variations in copy number is reflected in the overall error observed for the GalK assays. The wild type and moter region
6863
several of the mutant promoters were also transferred to a lambda vector and recombined into the chromosome in a single copy. The relative expression of mutants remained the same as that observed with the plasmid system. Mutagenesis. Hydroxylamine mutagenesis was carried out by the method of Busby et al. (7), with modifications. pWH54 DNA (2 ,ug) containing a 500-bp region of the araFG operon in 100 ,ul of 50 mM phosphate buffer was treated with 1 M hydroxylamine for 90 min at either 50 or 70°C. Aliquots of 50 ,ul were removed at 60 and 90 min. Samples were loaded directly onto a Bio-Rad P60 column, equilibrated with 10 mM Tris (pH 7.6), eluted, and ethanol precipitated. Samples were separated on 1% agarose, and the DNA was excised and isolated by electroelution. DNA was then digested with HindIlI-EcoRI; the fragments were isolated on 6% polyacrylamide gels, ligated with pKO1 (56), and transformed into WH75. Isolates were screened by plating on MacConkey-galactose plates containing ampicillin and arabinose. Sodium bisulfite mutagenesis was performed essentially as described previously (59). Single-stranded phage M13 DNA containing a 500-bp region of the araFG operon (10 ,ug of constructs cloned in opposite directions used in two reactions) was treated with 0.75 M sodium bisulfite and 0.5 mM hydroquinone for 30 min at 37°C in the dark. The reaction was terminated by loading the sample onto a Bio-Rad P60 column equilibrated with 10 mM Tris-HCl (pH 8.0) and primed with 50 pg of bovine serum albumin. The samples were brought to 0.2 M Tris-HCl (pH 9.0)-50 mM NaCl-2.0 mM EDTA and incubated at 37°C overnight. The DNA was then annealed to the primer, and a second DNA strand was synthesized by extension with Klenow polymerase. Mutagenesis with potassium permanganate was performed by the method of Myers et al. (47). pWH54 DNA (20 ,ug) was linearized by digestion with SmaI or EcoRI and made single stranded by digestion with 50 U of exonuclease III at room temperature overnight. The template was treated with 9 ,uM potassium permanganate at room temperature for 40 min and then ethanol precipitated. A second DNA strand was synthesized by extension of an oligonucleotide primer with reverse transcriptase at 42°C for 1 h. Oligonucleotide mutagenesis was performed by the method of Kunkel et al. (38). Oligonucleotides of 20 to 25 bases were synthesized on a Milligen model 7500 DNA synthesizer and purified by Nensorb (Dupont) column chromatography. Candidates were screened by sequencing (54), and the promoter restriction fragment was isolated by cutting replicative-form DNA from mutants. For candidates from all mutagenesis methods, the HindIII-KjpnI restriction fragment containing the promoter region was isolated and ligated into the HindIII-KjpnI large fragment of pWH54. Candidates were screened by transforming plasmids into WH75 and plating on MacConkey ampicillin-galactose-arabinose plates. An additional round of fragment isolation, cloning, and sequencing was performed to insure that no additional mutations were produced. Deletion mutagenesis. pWH54 DNA (10 ,ug) was digested with HindIII and then with 1 U of Bal 31 for 1 to 4 min at room temperature. Aliquots (20 ,ul) removed at 0.5, 1, 2, and 4 min were extracted with phenol once, extracted with ether three times, and then precipitated with ethanol. DNA samples were then ligated with phosphorylated HindIII linkers. The promoter-containing DNA fragments were isolated by digestion with HindIII-EcoRI, the DNA was separated by agarose gel electrophoresis, and the fragments were recovered on NA45 membranes (Schleicher & Schuell). DNA was
6864
HENDRICKSON ET AL.
J. BACTERIOL.
ligated into the 2.8-kb HindIII-EcoRI fragment of pWH54 and transformed into WH75. Transformants were screened by plating on MacConkey plates containing arabinose, galactose, and ampicillin. Plasmids of selected isolates were purified (53) and sequenced. Deletion of the region upstream of araFGl, from positions -130 to -505, in strain WH62 was accomplished by removing the TaqI-to-EcoRI fragment of pWH54, filling in the fragment ends with the Klenow fragment of DNA polymerase I, and religating the DNA. The region upstream of the CRP site was deleted by creating an EcoRI site by oligonucleotide mutagenesis (38), changing a T to a G at position -60. The resulting plasmid was cut with EcoRI, and the large fragment was ligated to produce a deletion from positions -59 to -505. Transcription in vitro. Each DNA (10 pLg) was digested with KjpnI-SnaBI and then ethanol precipitated. Each template (1 nmol) was then utilized for in vitro transcription as described previously (24). The DNA templates were added to buffer containing 20 mM Tris (pH 7.4), 100 mM KCl, 5 mM MgCl2, 0.25 mM EDTA, 1 mM dithiothreitol, 50 mM arabinose, 0.05 mM cAMP, and 50 mg of bovine serum albumin per ml. Reactions were brought to a final concentration of 0.5 ,tg of AraC protein per ml and 1.0 p,g of CRP per ml. Single-round transcription was initiated by adding 0.3 U of RNA polymerase (Promega or Epecentre Technologies) for 15 min (total volume, 21 ,ul) and then 2.0 pl of the following nucleotide mix: 2 mM GTP, 1 mM each CTP and UTP, 0.1 mM ATP, and 20 ,uCi [a-32P]ATP with 0.1 mg of heparin per ml. After incubation at 37°C for 30 min, reactions were stopped by adding an equal volume of a mixture containing 85% formamide, 40 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol. Samples were then heated to 90°C for 3 min and loaded onto a 10% acrylamide sequencing gel. For the association rate experiments, reactions were performed as described above, except that the nucleotide mix was added at time intervals after the addition of RNA polymerase. The samples were analyzed by loading the entire reaction mix on a 5% acrylamide denaturing gel. Quantitative data were obtained directly from the gel with a 3-scanning system (Ambis). 5'-Nucleotideidentification. y-32P-labeled nucleoside triphosphates were synthesized from kits (Promega) according to the manufacturer's instructions. Synthesis was monitored by thin-layer chromatography. Each labeled nucleotide was used in a separate transcription reaction with the other three unlabeled nucleotides, RNA polymerase, CRP-cAMP, and AraC-arabinose as described above. Reactions were analyzed on denaturing gels with an a-32P-labeled reaction as a standard. RNA sequence analysis. RNA for sequence analysis was produced from in vitro transcription reactions with pWH54 DNA as described above, except the template concentration was 4 nM (14.4 ng) in a reaction volume of 25 ,ul. Reactions were
multiple round, with addition of heparin after incuba-
tion for 30 min at 37°C. After completion, reactions were
purified over a Qiagen column
as
specified by the manufac-
turer of the column. For each sequence reaction and in the alkaline hydrolysis control reaction, 5,000 cpm of transcript was used. All reactions were done as specified by Bethesda Research Laboratories, Inc. Optimal conditions for araFGH RNA reactions were 1 U of RNase T1, 0.6 U of RNase U2, and 1.2 U of RNase CL3.
TABLE 1. Deletions of the araFGH promoter Strain
Mutatione
WH54 Deletions from upstream WH62 WH608 Deletions from downstream WH594 WH606 WH593
WH604 WH605 WH603 WH592 WH601 WHS91 WH600 WH589 WH607 WH602 WH598 WH599
Relative GalK activityb +Arabinose
-Arabinose
Wild type
100
1.5
-130 -59
48 3
1-2 1-2
+8 +4 +3 +2 -5 -6 -7 -10 -12
128 109 90 134 88 114 120 61
-
-21 -32 -35 -51 -59 -64
48 6 1-2 1-2 1-2 1-2 1-2
1-2
1-2 3 1-2
Vol. 174, No. 21
0021-9193/92/216862-10$02.00/0 Copyright © 1992, American Society for Microbiology
Sequence Elements in the Escherichia coli araFGH Promoter WILLIAM HENDRICKSON,* CLAIRE FLAHERTY,t AND LISA MOLZ Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, P. O. Box 6998, Chicago, Illinois 60680 Received 1 May 1992/Accepted 27 August 1992
The Eschcrichia coli araFGH operon codes for proteins involved in the L-arabinose high-affinity transport system. Transcriptional regulation of the operon was studied by creating point mutations and deletions in the control region cloned into a GalK expression vector. The transcription start site was confirmed by RNA sequencing of transcripts. The sequences essential for polymerase function were localized by deletions and point mutations. Surprisingly, only a weak -10 consensus sequence, and no -35 sequence is required. Mutation of a guanosine at position -12 greatly reduced promoter activity, which suggests important polymerase interactions with DNA between the usual -10 and -35 positions. A double mutation toward the consensus in the -10 region was required to create a promoter capable of significant AraC-independent transcription. These results show that the araFGH promoter structure is similar to that of the galPI promoter and is substantially different from that of the araBAD promoter. The effects of 11 mutations within the DNA region thought to bind the cyclic AMP receptor protein correlate well with the CRP consensus binding sequence and confirm that this region is responsible for cyclic AMP regulation. Deletion of the AraC binding site nearest the promoter, araFGl, eliminates arabinose regulation, whereas deletion of the upstream AraC binding site, araFG2, has only a slight effect on promoter activity.
associated and have regions of strong sequence similarity with other proteins involved in sugar transport (31, 58). Both transport operons, as well as the operon, araBAD, for the catabolic enzymes are regulated at the initiation of transcription by the intracellular levels of cyclic AMP (cAMP) and L-arabinose (15, 22, 37, 66). The cAMP receptor protein (CRP) is responsible for catabolic regulation in response to cAMP, and the AraC protein responds to L-arabinose. Both of these activators bind adjacent sites of the promoters just upstream from the RNA polymerase binding region. At the araFGH promoter, however, the orientation of the sites is reversed as compared with those of the other ara promoters; the CRP site is adjacent to that for RNA polymerase, and AraC binds upstream of CRP (Fig. 1) (29), whereas at araBAD AraC binds adjacent to polymerase and CRP binds upstream (48). These promoter structures help explain some of the differences between the ara promoters with regard to regulation. araFGH is highly sensitive to catabolite repression and is not capable of significant transcription in the absence of CRP either in vivo or in vitro (29, 37). On the other hand, araBAD is only moderately regulated by CRP and can be substantially induced by arabinose-AraC alone. Transcription of the araBAD operon is repressed by the action of AraC protein in a looped DNA complex with a single molecule of AraC bound to an upstream site, araO2, and to the inducer site, araI (33, 40, 43). CRP appears to activate transcription directly and also indirectly by breaking the repression loop (23, 24, 39, 41, 61). It has been suggested that araFGH is also repressed by AraC protein in the absence of arabinose (37). The araBAD promoter has recognizable -10 and -35 hexamers. Mutations that decrease transcription, or increase transcription in the absence of activators, have been found at both sites (10, 20, 23). In the study reported here, we performed the first genetic analysis of the araFGH promoter. The promoter region was fused to the galactokinase gene to isolate mutants with decreased or increased promoter function. The mutants were characterized with respect to the induced and basal levels of expression. These
The RNA polymerase binding sites of many promoters have been studied in detail. A consensus sequence for recognition by RNA polymerase c7o includes two conserved hexanucleotide sequences located 10 and 35 bp upstream from the transcription start site (25, 26, 46, 67). Additional sequences between and upstream of these elements are weakly conserved but can significantly affect promoter strength (2, 50). A model of polymerase recognition based on genetic experiments showing interactions of different amino acid motifs within the cr subunit with specific base pairs of the -10 and -35 regions has been developed (18, 60, 65). Our present knowledge of promoter structure is based primarily on the analysis of strong promoters that do not require accessory proteins. Promoters requiring activators frequently have a -35 region with poor similarity to the consensus (25). It is not clear, however, whether these promoters are simply characterized by a poor polymerase recognition site or whether they contain unique recognition elements. We are studying the Escherichia coli promoters that regulate genes involved in arabinose metabolism to understand structure-function relationships of promoters that require multiple activators for transcription. T`wo distinct systems are capable of active transport of the sugar L-arabinose in E. coli. A low-affinity, high-capacity symporter system is the product of the araE operon (6, 36, 62). At present only one protein is known to be encoded by this operon. A high-affinity system is encoded by the araFGH operon located at 41.9 min (2,000 bp) on the E. cdli map (9, 27, 37, 58). The product of the araF gene, L-arabinose binding protein, is located in the periplasm and is required for high-affinity transport (30, 55), and two additional genes of unknown function, araG and araH, have been identified. Both araG and araH products are membrane *
Corresponding author.
t Present address: Department of Anatomy and Neuroscience, Pennsylvania State University College of Medicine, Hershey, PA 17033. 6862
araFGH PROMOTER
VOL. 174, 1992 -155
_ AraC
-80
41
araF
AraC CRP
RNA pol.
araFGl araFG2 FIG. 1. Control region of the araFGH promoter. Binding sites for AraC protein, CRP, and RNA polymerase are shown. The distance from the transcription start site (arrow) to the center of each activator protein is given above the diagram. In plasmid pWH54, the 505-bp DNA fragment containing the control region is fused to galK via a HindIII linker at position +90.
mutants allowed
us to clearly identify the protein binding sites and show that the RNA polymerase binding site is much different than that suggested for the araBAD promoter; only minimal polymerase recognition elements are present, and an important recognition element lies adjacent to the -10 region.
MATERIALS AND METHODS General procedures. pWH54 contains the araFGH proon a 500-bp HindIII-EcoRI fragment (Fig. 1) (29). All plasmids were introduced by transformation into strain WH75 (F- leu Alac-74 galK thi araB, Strr), which is araC+ and araBAD. WH75 was constructed by P1 transduction of the polar araB mutation of SH313 into E. coli K-12 strain SH2. Gel electrophoresis band shift assays were performed as described previously (28). A 235-bp restriction fragment of pWH54 was end labeled with 32P and used in the gel-shift assay. All general DNA manipulations, restriction digestions, ligations, and DNA transformations were as described previously (53, 56). Galactokinase assays. Cultures (20 ml) were grown in M9 medium (53) plus 4 p,g of thiamine per ml, 0.2% Casamino Acids, and 20 pg of ampicillin per ml at 37°C to an A650 of 0.6. Galactokinase assays were performed on toluenetreated cells by the method of McKenney et al. (44). In this assay the production of [14C]galactose phosphate is assayed by its retention on DEAE filters (Whatman DE81). Units are millimoles of galactose phosphate produced per minute per milliliter of culture (A650, 1). Determinations were made in triplicate for each culture, with at least two separate cultures utilized for each strain. Cells were induced by adding 0.2% arabinose to the cultures for at least four generations. Parallel ampicillinase measurements by the nitroceffin assay (63) were performed on a portion of the toluenetreated cells of each culture to insure that the plasmid copy number remained constant. The reaction of ampicillin from the permeabilized cells with nitroceffin was measured by the increase inA492. The copy number of the pKO1 system used for these studies has been reported to vary when actively transcribed genes are present (1). We carefully tested the constructs and found that transcription from the araFGH promoter is not sufficient to alter the copy number. All of the mutants had the same copy number (±10%) as the wild type within the error of the assay. Occasional cultures with significant variations in copy number were discarded. Since multiple cultures were done for each mutant, the error due to random variations in copy number is reflected in the overall error observed for the GalK assays. The wild type and moter region
6863
several of the mutant promoters were also transferred to a lambda vector and recombined into the chromosome in a single copy. The relative expression of mutants remained the same as that observed with the plasmid system. Mutagenesis. Hydroxylamine mutagenesis was carried out by the method of Busby et al. (7), with modifications. pWH54 DNA (2 ,ug) containing a 500-bp region of the araFG operon in 100 ,ul of 50 mM phosphate buffer was treated with 1 M hydroxylamine for 90 min at either 50 or 70°C. Aliquots of 50 ,ul were removed at 60 and 90 min. Samples were loaded directly onto a Bio-Rad P60 column, equilibrated with 10 mM Tris (pH 7.6), eluted, and ethanol precipitated. Samples were separated on 1% agarose, and the DNA was excised and isolated by electroelution. DNA was then digested with HindIlI-EcoRI; the fragments were isolated on 6% polyacrylamide gels, ligated with pKO1 (56), and transformed into WH75. Isolates were screened by plating on MacConkey-galactose plates containing ampicillin and arabinose. Sodium bisulfite mutagenesis was performed essentially as described previously (59). Single-stranded phage M13 DNA containing a 500-bp region of the araFG operon (10 ,ug of constructs cloned in opposite directions used in two reactions) was treated with 0.75 M sodium bisulfite and 0.5 mM hydroquinone for 30 min at 37°C in the dark. The reaction was terminated by loading the sample onto a Bio-Rad P60 column equilibrated with 10 mM Tris-HCl (pH 8.0) and primed with 50 pg of bovine serum albumin. The samples were brought to 0.2 M Tris-HCl (pH 9.0)-50 mM NaCl-2.0 mM EDTA and incubated at 37°C overnight. The DNA was then annealed to the primer, and a second DNA strand was synthesized by extension with Klenow polymerase. Mutagenesis with potassium permanganate was performed by the method of Myers et al. (47). pWH54 DNA (20 ,ug) was linearized by digestion with SmaI or EcoRI and made single stranded by digestion with 50 U of exonuclease III at room temperature overnight. The template was treated with 9 ,uM potassium permanganate at room temperature for 40 min and then ethanol precipitated. A second DNA strand was synthesized by extension of an oligonucleotide primer with reverse transcriptase at 42°C for 1 h. Oligonucleotide mutagenesis was performed by the method of Kunkel et al. (38). Oligonucleotides of 20 to 25 bases were synthesized on a Milligen model 7500 DNA synthesizer and purified by Nensorb (Dupont) column chromatography. Candidates were screened by sequencing (54), and the promoter restriction fragment was isolated by cutting replicative-form DNA from mutants. For candidates from all mutagenesis methods, the HindIII-KjpnI restriction fragment containing the promoter region was isolated and ligated into the HindIII-KjpnI large fragment of pWH54. Candidates were screened by transforming plasmids into WH75 and plating on MacConkey ampicillin-galactose-arabinose plates. An additional round of fragment isolation, cloning, and sequencing was performed to insure that no additional mutations were produced. Deletion mutagenesis. pWH54 DNA (10 ,ug) was digested with HindIII and then with 1 U of Bal 31 for 1 to 4 min at room temperature. Aliquots (20 ,ul) removed at 0.5, 1, 2, and 4 min were extracted with phenol once, extracted with ether three times, and then precipitated with ethanol. DNA samples were then ligated with phosphorylated HindIII linkers. The promoter-containing DNA fragments were isolated by digestion with HindIII-EcoRI, the DNA was separated by agarose gel electrophoresis, and the fragments were recovered on NA45 membranes (Schleicher & Schuell). DNA was
6864
HENDRICKSON ET AL.
J. BACTERIOL.
ligated into the 2.8-kb HindIII-EcoRI fragment of pWH54 and transformed into WH75. Transformants were screened by plating on MacConkey plates containing arabinose, galactose, and ampicillin. Plasmids of selected isolates were purified (53) and sequenced. Deletion of the region upstream of araFGl, from positions -130 to -505, in strain WH62 was accomplished by removing the TaqI-to-EcoRI fragment of pWH54, filling in the fragment ends with the Klenow fragment of DNA polymerase I, and religating the DNA. The region upstream of the CRP site was deleted by creating an EcoRI site by oligonucleotide mutagenesis (38), changing a T to a G at position -60. The resulting plasmid was cut with EcoRI, and the large fragment was ligated to produce a deletion from positions -59 to -505. Transcription in vitro. Each DNA (10 pLg) was digested with KjpnI-SnaBI and then ethanol precipitated. Each template (1 nmol) was then utilized for in vitro transcription as described previously (24). The DNA templates were added to buffer containing 20 mM Tris (pH 7.4), 100 mM KCl, 5 mM MgCl2, 0.25 mM EDTA, 1 mM dithiothreitol, 50 mM arabinose, 0.05 mM cAMP, and 50 mg of bovine serum albumin per ml. Reactions were brought to a final concentration of 0.5 ,tg of AraC protein per ml and 1.0 p,g of CRP per ml. Single-round transcription was initiated by adding 0.3 U of RNA polymerase (Promega or Epecentre Technologies) for 15 min (total volume, 21 ,ul) and then 2.0 pl of the following nucleotide mix: 2 mM GTP, 1 mM each CTP and UTP, 0.1 mM ATP, and 20 ,uCi [a-32P]ATP with 0.1 mg of heparin per ml. After incubation at 37°C for 30 min, reactions were stopped by adding an equal volume of a mixture containing 85% formamide, 40 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol. Samples were then heated to 90°C for 3 min and loaded onto a 10% acrylamide sequencing gel. For the association rate experiments, reactions were performed as described above, except that the nucleotide mix was added at time intervals after the addition of RNA polymerase. The samples were analyzed by loading the entire reaction mix on a 5% acrylamide denaturing gel. Quantitative data were obtained directly from the gel with a 3-scanning system (Ambis). 5'-Nucleotideidentification. y-32P-labeled nucleoside triphosphates were synthesized from kits (Promega) according to the manufacturer's instructions. Synthesis was monitored by thin-layer chromatography. Each labeled nucleotide was used in a separate transcription reaction with the other three unlabeled nucleotides, RNA polymerase, CRP-cAMP, and AraC-arabinose as described above. Reactions were analyzed on denaturing gels with an a-32P-labeled reaction as a standard. RNA sequence analysis. RNA for sequence analysis was produced from in vitro transcription reactions with pWH54 DNA as described above, except the template concentration was 4 nM (14.4 ng) in a reaction volume of 25 ,ul. Reactions were
multiple round, with addition of heparin after incuba-
tion for 30 min at 37°C. After completion, reactions were
purified over a Qiagen column
as
specified by the manufac-
turer of the column. For each sequence reaction and in the alkaline hydrolysis control reaction, 5,000 cpm of transcript was used. All reactions were done as specified by Bethesda Research Laboratories, Inc. Optimal conditions for araFGH RNA reactions were 1 U of RNase T1, 0.6 U of RNase U2, and 1.2 U of RNase CL3.
TABLE 1. Deletions of the araFGH promoter Strain
Mutatione
WH54 Deletions from upstream WH62 WH608 Deletions from downstream WH594 WH606 WH593
WH604 WH605 WH603 WH592 WH601 WHS91 WH600 WH589 WH607 WH602 WH598 WH599
Relative GalK activityb +Arabinose
-Arabinose
Wild type
100
1.5
-130 -59
48 3
1-2 1-2
+8 +4 +3 +2 -5 -6 -7 -10 -12
128 109 90 134 88 114 120 61
-
-21 -32 -35 -51 -59 -64
48 6 1-2 1-2 1-2 1-2 1-2
1-2
1-2 3 1-2
