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Nucleic Acids Research Volume 9 Number 21 1981 Nucleic Acids Research The nucleotide sequence of the promoter and the amino-terminal region of alkaline phosphatase structural gene (phoA) of Escherichia coli Yasuhiro Kikuchi, Koji Yoda, Makari Yamasaki and Gakuzo Tamura Department of Agricultural Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Received 21 September 1981 ABSTRACT The promoter and the amino-termianl region of phoA, the structural gene for alkaline phosphatase of Escherichia coli K12, was cloned by using a pro- moter cloning vector pMC1403. The nucleotide sequence of the cloned fragment has been determined. A sequence encoding the amino-terminal portion of mature alkaline phosphatase is found and it is preceded by a sequence encoding the signal peptide. The signal peptide consists of 21 amino acids; Met-Lys-Gln- Ser-Thr-Ile-Ala-Leu-Ala-Leu-Leu-Pro-Leu-Leu-Phe-Thr-Pro-Val-Thr-Lys-Ala. The translation initiation codon is GUG, which is preceded by the Shine- Dalgarno sequence GGAG. Upstream to these sequences, there is a typical pro- caryotic promoter, TATAGTC for the Pribnow box. Around the Pribnow box, there are several dyad symmetrical sequences, which may probably be concerned with the 'regulation of this gene. INTRODUCTION Alkaline phosphatase (EC 3.1.3.1.) of E.coli is synthesized under low phosphate conditinos (1) and is secreted across the inner membrane to the periplasmic space (2). Its structural gene phoA is located at 8.5 min of the E.coli genetic map (3). The regulatory mechanism of expression of phoA is complex and not well understood. A number of genes, including phoB (4), phoM (5), phoR (6), phoS, phoT (7) and perA (8), have been identified, which af- fect the expression of phoA. Gene products of phoB, phoM and phoR especially regulate transcription of phoA and may interact directly with some specific nucleotide sequence(s) around the phoA promoter. Like other secreted proteins, alkaline phosphatase is synthesized as a precursor form which has a signal peptide at the amino-terminal (9). The sig- nal peptide is believed to play an important role in the initiation step(s) of protein-translocation across the membrane and is cleaved off, during or soon after the secretion (10,11,12,13). The amino acid sequence of the signal peptide of alkaline phosphatase has been only partially known (14). The promoter region of phoA is interesting because of its complex regu- latory mechanism of expression and because of the existence of the signal ©) IRL Press Umited, 1 Falconberg Court, London W1V 5FG, U.K. 5671 Nucleic Acids Research peptide at the amino-terminal. Previously, we have cloned the entire phoA gene on plasmid vectors (15). We report here the subcloning of the phoA pro- moter region and the determination of its nucleotide sequence. MATERIALS AND METHODS Bacterial strains and plasmids. E.coli strain MC1061 (AlacZ) and plasmid pMC1403 were kindly provided by Dr.M.J.Casadaban (16). Construction of phoA plasmid pKI-2 has been described previously (15). Media and transformation. Phosphate limited "media 121" (17) containing 5-bromo-4-chloro-3-indolyl-D-galactoside (SIGMA) (18) was used for selecting Lac transformants under low phosphate conditions. Transformation with plasmid DNA was carried out as described by Cohen et al (19). Enzymes. All enzymes used were purchased from commercial sources. Reac- tions were carried out under conditions described by suppliers. Purification of plasmid DNA. For small scale (10 ml) preparations, we adopted the "mini prep" described by Klein et al (20). For large scale (2000 ml) preparations, covalently closed circular plasmid DNA was purified from cleared lysate by using hydroxyapatite column chromatography (21). Cloning of the phoA promoter region. pKI-2 and pMC1403 were mixed digested with EcoRI and ligated with T4 DNA ligase. MC1061 was transformed with this DNA mixture. Transformants, which were resistant to ampicillin and showed Lac+ phenotypes under low phosphate conditions, were isolated and their plasmids were analysed. pYK171, one of such plasmids, was partially digested with HinfI to the extent that most of the plasmid molecules could be cleaved at only one site. Linearized molecules were purified by agarose gel electrophoresis. HinfI staggered ends were repaired with E.coli DNA polymerase I (large subunit) in the presence of four d-NTPs. Repaired mole- cules were digested with SmaI and circularized with T4 DNA ligase. Transfor- mants of MCl061, which were ampicillin resistant and showed Lac phenotypes under low phosphate conditions, were selected and further analysed. Purification of DNA fragments and restriction mapping. DNA fragments were isolated and purified by polyacrylamide gel electrophoresis as described by Maxam and Gilbert (22). A restriction map was established by sizing the DNA fragments digested with various restriction enzymes on polyacrylamide gels containing ethidium bromide. HinfI digested fragments of pBR322 were used as molecular weight markers (23). DNA sequencing. DNA fragments were 5' end labeled with [y- P] ATP and sequenced as described by Maxam and Gilbert (22). 5672 Nucleic Acids Research RESULTS Cloning of the _hoA promoter region. We have previously constructed pKI- 2 which has the entire phoA gene cloned on pBR322 (15). Subcloning studies have revealed that the phoA gene is on the 2.7 kb (kilo bases) fragment be- tween HindME site and XhoI site and that the phoA promoter is on the 1.25 kb fragment between HindME site and EcoRI site (Kikuchi et al. Abstr. Jpn. Bio- chem. Soc. Meet. 1980, 3Q, p.11 and Kikuchi et al. in preparation). We subcloned the 1.28 kb EcoRI fragment of pKI-2 into the EcoRI site of pMC1403, as described in materials and methods section and as shown in Fig.l. Transformants with pYK171 s,howed Lac+ phenotype, only under low phosphate conditions. There are four HinfI sites on the 1.25 kb fragment between HindMI site and EcoRI site of pYK171. Fragments of various length between SmaI site and one of the HinfI sites were deleted as described in materials and methods section and as shown in Fig.l. Several kinds of plasmids smaller than pYK171 were obtained containing the phoA promoter. Among those plasmids, pYKl90 had the 520 bp (base pairs) fragment between BamHI site and HindIm site. Trans- formants with pYKl90 showed Lac phenotype, only under low phosphate condi- tions. Z 1.25 E 0.35 4.02 JAVA~N~E1.10 IPKI-21 YcOJ 9.90 IwRI Ligation 0.52 EB1.25~~ ~ ~ ~ ~ ~ ~ E luutl 4 d-ETPs aI T4 ligase UWa2 1 PYK171 partial repair l PYKi90O 9.90_ 9.90 Fig.l Cloning of the phoA Promoter Region. (i) pBR322 DNA, () pMC1403 DNA, (Ei) E.coli DNA, () the phoA promoter. Cleavage sites of restric- tion enzymes; (E) EcoRI, (H) Hind4 , (X) XhoI, (S) SmnaI and (B) BamHI. Lengths of fragments are indicated in kilo bases. 5673 Nucleic Acids Research Nucleotide sequence of the 520 bp fragment. pYK190 was digested with BamHI and HindMEI. The 520 bp fragment was purified and the restriction map was established with AluI, Haelf, HhaI, HinfI and HpaU . Fig.2 shows the map and the strategy we adopted for sequencing the fragment. Fig.3 reports the nucleotide sequence we have determined. DISCUSSION We have cloned the promoter region of phoA by using a promoter cloning vector pMC1403 (16). Several recombinant plasmids, including pYK171 and pYK 190, that carried the phoA-lacZ fusion genes, were obtained. The transformants showed Lac+ phenotypes, only under low phosphate conditions. Details on the expression of the fused genes and on the translocation of the products will be described elsewhere. We have reported here the nucleotide sequence of the 520 bp fragment of pYK190, that contains the promoter region of phoA. Within the sequence shown in Fig.3, sequence GGGGATC (nucleotides 514- 520) originated from vector pMC1403. The other 514 bp sequence originated from E.coli chromosome. Sequence GACT (nucleotides 510-513) is generated by the repair of HinfI staggered end and it is ligated to the blunt end of SmaI ~~~~~~~~~~~~~~~Z 4. (2) - _1--- (3) 0 (4) O 100 200 300 400 50 bp Fig.2 Strategy for Sequencing the 520 bp HindM-BanHI Fragment of pYK190. (O ) indicates the position of "P label at 5' end. Dotted lines indicate re- gions not sequenced. (1) 5'-labeled and cleaved with HaeM. (2) Cleaved with HaeI, 5'-labeled and separated into single strands. (3) Cleaved with AluI, 5'-labeled and cleaved with HinfI. (4) Cleaved with HinfI, 5'-labeled and cleaved with HhaI. 5674 Nucleic Acids Research 100 * * * * * * * * * * 5' - AZ4uIGludIZGVaZTPhrAZaAbtLeaugA5AenMtAZaGZL4BanAspGZnGZnArgLeuIZeA8pGZnVaZGZuGZLJAZarLeu2lVG1uVaZLya 200 * * * * * * * * *_ * P?oApAAaSerIZePOAapAspAsp2pGlueuLeuArgA8pS¶yzVaZLyaLyeLeuLeuLyeHi.eProArgGIn 300 * *m _ T?ATGTATTTGTACATGGAGAAAATAAGTGAAACAAAGCACT&TTG PB MetLyeGlnSerThzrIZe SD -20 400 * * * * * * * * * * CACTGGCACTCTTACCG r&CTGTTTACCCCTGTGACAAGCCCGGACACCAGAAATGCCTGTTCTGGAAAACCGGGCTGCTCAGGGCGATATTACTGC _G_Ga__CGTGAGA&TGGCAATGACMATGGGGACGTrI"ZcGGGcCTGTGGTCTTrACGGACAAGAcCCTTTGGCCCGAcGAGTCCCGCTATAATGAcG AZaLeuAZaLeuLeuProLeLuPerPro lroVyaZlhrLyaAZuft2elhr~VGZLuetPoVGLuZuAsn4rgAZaA ZaGZnGZyAspIZelhrA Za -10 -1 1' +1 10 500 * * * * * * * * * * ACCCGGCGGTGCTCGlCGTTTAACGGGTGATCAGACTGCCGCTCTGCGTGATTCTCTTAGCGATAACCTGCAAAATATTATTTTGCTGATrGGCGAT TGGGCCGCCACGAGCGGCAAATTGC GACTAACCGCTA PFoGZyGZyAlaA4gArgLeulrGZyApGZnThrAZaAZaLeuArgAspSerLeuSerAspLysProAZaLyseAnIZeIZeLeuLeuIZeGZyAsp 20 30 40 50 * * GGG&TGGGGGACTGGGGATCC CCCTACCCCCTGACCCCTAG GlyMetGlyAap Fig.3 The Nucleotide Sequence of the 520 bp Fragment. (I ) and (in) indi- cate dyad symmetrical sequences
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