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Autoregulation of Escherichia Coli Purr Requires Two Control Sites Downstream of the Promotert RONDA J

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Autoregulation of Escherichia Coli Purr Requires Two Control Sites Downstream of the Promotert RONDA J JOURNAL OF BACTERIOLOGY, Oct. 1990, p. 5758-5766 Vol. 172, No. 10 0021-9193/90/105758-09$02.00/0 Copyright © 1990, American Society for Microbiology Autoregulation of Escherichia coli purR Requires Two Control Sites Downstream of the Promotert RONDA J. ROLFES AND HOWARD ZALKIN* Department ofBiochemistry, Purdue University, West Lafayette, Indiana 47907 Received 20 April 1990/Accepted 13 July 1990 The expression of Escherichia coli purR, which encodes the pur regulon repressor protein, is autoregulated. Autoregulation at the level of transcription requires two operator sites, designated purRol and purRo2 (O1 and 02). Operator 01 is in the region of DNA between the transcription start site and the site for translation initiation, and 02 is in the protein-coding region. The repressor. protein binds noncooperatively to 01 with a sixfold-higher affinity than to 02, and saturation of 01 by the repressor precedes saturation of 02. Both Oi and 02 function in the two- to threefold autoregulation in yto; as determined by measurement of 0-galactosidase and mRNA from purR-lacZ translational fusions. Of all the genes thus far known to be regulated by the Pur repressor, only purR employs a two-operator mechanism. In Escherichia coli, the genes encoding the enzymes for E. coli purR. Our work supports and extends their analyses the de novo synthesis of IMP are arranged as individual loci and gives a more complete picture of the autoregulation of and small polycistronic operons. The gene organization and purR. map locations (in minutes) are as follows (2, 33): purB, 25.2; purHD, 90.3; purEK, 12.2; cvp purF dedF (11, 37), 50.0; MATERIALS AND METHODS purMN, 53.5; purC, 53.3; purL, 55.2. In addition, guaBA at Bacterial strains and plasmids. Strains and piasmids are min 53.9 is required for the two-step conversion of IMP, to described in Table 1. GMP, and purA at min 95.0 and purB are required for Media. Minimal growth medium contained salts (50), 0.5% conversion of IMP.to AMP. The addition of exogenous glucose, 0.2% acid casein hydrolysate, and 2 utg of thiamine purines to the growth medium causes repression of all genes per ml. Adenine (100 ,ug/ml) was. added to repress purine in the pathway (15, 36). However, the AMP and GMP nucleotide synthesis. Extracts containing Pur repressor were branches.appear to be under separate regulation from the prepared from cells grown in rich medium containing 2% pathway leading to IMP (15, 54). Genes for the pathway to tryptone, 1% yeast extract, and 0.5% NaCl. L agar contain- IMP, except.for purB (23), are coregulated by the purR- ing 5-bromo-4-chloro-3-indoyl-i-D-galactoside was used to enc.oded repressor (23, 34) and a corepressor that is a small visualize j-galactosidase molecule (25). These genes constitute the E. coli pur regu- activity. from purR-iacZ fusions in lon. Gene purR encoding the pur regulon repressor has been cells on plates. For plasmid selection, antibiotics were added cloned (42)-and sequenced, and operator binding sites have to the following concentrations: ampicillin, 100 ,ug/ml; spec- been identified (29, 42). Each of the coregulated pur genes tinomycin, 50 p.g/ml. has a 16-base-pair (bp) conserved operator sequence that is -Plasmid conmstructions. A series of plasmids (Table 1) was located in the promoter region (1, lla, 32, 43, 45, 47, 47a, 48, produced in the construction of pPR1006, the source of 51; K. A. Flannigan, S. H. Hennigan, H. H. Vogelbacker, J. fragments for binding assays. A 3.2-kilobase BalI-to-PstI S. Gots, and J. M. Smith, Mol. Microbiol., in press; A. A. fragment (Fig. 1A) from purR+ plasmid pPR1003 was sub- Tiedeman, D. J. DeMarini, J. Parker, and J. M. Smith, cloned into the HincIl and PstI. sites of pUC119, yielding submitted- for publication). Mutational analysis (41) and plasmid pPR1004. The. 3' noncoding sequences were re- DNase I footprinting (23, 42) have established that the Pur moved by deleting sequences between the PstI site and the repressor binds to these operator sequences for the negative HpaI site located at position 1266 relative to the transcrip- regulation ofpur regulon gene expression (23, 34). tion start site of purR. The resulting plasmid, pPR1004-2, Gene purR contains two operatorlike sequences located contained the entire coding sequence ofpurR and 136 of 155 downstream of the promoter (42). In this report, we show nucleotides of the 5' transcribed but untranslated sequence that purR is autoregulated and that the operatorlike se- cloned in the opposite direction to the lac promoter. An quences purRo1 and purRo2 (01 and 02) are authentic NdeI site was constructed at the initiating ATG codon in control sites. The mechanism of autoregulation involves the plasmid pPR1004-2 by oligonucleotide-directed mutagenesis independent binding of Pur repressor toO1 and 02- Operator (31) to produce pPR1005. Plasmid pPR1006 is a derivative of site 02, located within the purR coding sequence, binds the pPR1005 in which most of the coding sequence was removed repressor in vitro with a sixfold lower affinity than 01 and yet by deleting from an internal HincII site at nucleotide 321 to makes an important contribution to in vivo autoregulation the downstream polylinker SphI site. under the conditions studies. During the preparation of this A series of plasmids was produced in the construction of manuscript, Meng et al. (34) reported the purR-lacZ fusions and the mutagenesis of 01 and 02. Plas- autoregulation of mid pPR2000 contains the promoter region and was con- structed by cloning the XhoI (position -245)-to-BglI (posi- tion 258) fragment (Fig. 1A) from pPR1002 into the Hincll * Corresponding author. site of pUC119. A purR-IacZ fusion was constructed in two t Journal paper no. 12559 from the Purdue University Agricul- steps by subcloning purR' DNA from pPR2000 into the tural Experiment Station. low-copy-number vector pGB2 by using EcoRI and HindIII 5758 VOL. 172, 1990 AUTOREGULATION OF purR 5759 TABLE 1. E. coli strains and plasmids polylinker sites. A Pstl fragment containing lac'Z from pMC1871 was ligated downstream of the purR sequences in Strain or Description Reference or plasmid Dsrpinsource the PstI site. The fusion gene created at this step (Fig. IB) contained 35 codons of purR and 13 codons from a poly- E. coli linkerjoined to codon 8 of lacZ. This purR-lacZ translational MC4100 A(argF-lac)169 4 R320 MC4100purR300 41 fusion was under the same controls as the purR gene. The CJ236 dut-J ung-J 31 copy number of this plasmid is expected to be maintained between four and six copies per cell (21). Plasmids Substitutions were made in the operators 01 and 02 by pPR1002 3.8-kilobase purR+ PstI fragment in 42 oligonucleotide-directed mutagenesis with pPR2000 as the pMS421 source of single-stranded DNA. The purR-lacZ fusions were pPR1003 Opposite orientation of PstI fragment 42 created by using the same cloning scheme described for the in pPR1002 pPR1004 purR BalI-to-Pstl fragment in This work wild-type fusion. The plasmid designations for the purR-lacZ pUC119 containing 01-02, 0102-7 and 01-02- are pPR2004, pPR1004-2 purR BalI-to-HpaI fragment in This work pPR2005, and pPR2006, respectively. pUC119 Oligonucleotide-directed mutagenesis. Mutagenesis was pPR1005 pPR1004-2 with NdeI mutation This work performed by the method of Kunkel (31) with the Muta-Gene pPR1006 purR' BaIl-to-HincII fragment in This work Phagemid in vitro mutagenesis kit from Bio-Rad Laborato- pUC119 ries. An NdeI site was created in pPR1004-2 by using the pPR2000 purR' XhoI-to-BglI fragment in This work pUC119 oligonucleotide 5'-TGGAGTGAAATCAIATGGCAACAA pPR2000-1 pPR2000 with 01- This work TAAAAG-3' (mismatches are underlined). The operator pPR2000-2 pPR2000 with 02- This work mutations were made in pPR2000 by using the oligonucleo- pPR2000-3 pPR2000-2 with 01- This work tides 5'-GTAAAGGCAAACACATACCTTGCGATTTTG- pPR2002 Wild-type purR-lacZ fusion (0102) This work 3' and 5'-GAAACGAGCAAACGTCAGTACTACAACT pPR2004 purR-lacZ (01702) This work GT-3' to mutate 01 and 02, respectively (Fig. 1). All pPR2005 purR-lacZ (0102) This work mutations were verified by dideoxy sequence analysis (44). pPR2006 purR-lacZ (017020 ) This work Assay of I-galactosidase. Cells were grown to the pGB2 Low-copy-number vector, Spcr 6 midlog pMS421 lacIq gene in pGB2, Spcr 42, M. Sus- phase in minimal medium with and without adenine. The kinda P-galactosidase activity of purR-lacZ fusions was deter- pUC119 High-copy-number phagemid, Ampr 49 mined by the Miller assay (35). Since purR andpurR+ strains pMC1871 lac'Z cassette plasmid 46 were each grown with and without added purine, it was possible to calculate repression by two methods (Table 2). In a University of Southern Los Angeles. California, every case, the values calculated by the two methods agreed closely and supported the reliability of these measurements of repression. Repressor-operator binding. Protein extracts were pre- pared as described previously (42) from 20-ml cell cultures A. BgII HincI I HpaI XhoI (-245) +1 BalI (20) (258) (323) (1266) PstI \ 01 B. -MC-GCC-GGA-CCT-GCA-GGT-CGA-CGG-ATC-CGG-GGA-AU7-CCC-GGG-GAT-CCC-GTC- Asn - Ala3. Gly- Pro - Ala - Gly - Arg-Arg- lie - Arg - Gly - Ile - Pro - Gly - Asp- Pro; Val C. °1 MlGlR_AI 02 G A _|CNA C 02- GA_s3 WCAGTAC FIG. 1. (A) Schematic representation of the purR gene and surrounding region. The drawing is not to scale so that details will show. The positions of several restriction sites with respect to the transcription start site (arrow at +1) are shown in parentheses.
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