Regulation of Escherichia coli purA by purine repressor, one component of a dual control mechanism

Abstract

Escherichia colipurA encodes adenylosuccinate synthetase, one of two enzymes required for synthesis of AMP from IMP. purA is subject to two-to threefold regulation by purR and about twofold regulation by a purR-independent mechanism. The 5'-flanking region ofpurA confers purR-dependent transcriptional regulation ofpurA but not the purR-independent regulation. Two operator sites in the 5'-flanking region which bind purine repressor in vitro and are required for in vivo regulation were identified. The purR-independent regulation may be posttranscriptional. It is now established that all transcription units involved in de novo synthesis of purine nucleotides, nine pur operons, as well as purR itself and guaBA, are subject to purR control.Adenylosuccinate synthetase (EC 6.3.2.6), the product of the purA gene in Escherichia coli, catalyzes the first of two reactions from the IMP branch point to AMP in the de novo purine nucleotide biosynthetic pathway (21). The second reaction to AMP is catalyzed by adenylosuccinate lyase (EC 4.2.2.2), the product of purB. Genetically, purA is located at min 95 on the E. coli chromosome and is unlinked to otherpur regulon genes (1). The purA gene has been cloned and sequenced (32), and its expression is modulated by the levels of purines in the growth medium (31). On the basis of assays of adenylosuccinate synthetase used to monitor purA expression and 0-galactosidase for purB-lacZ expression, it was concluded that purA and purB are regulated separately (31). More recent information indicates that purB expression is coregulated with those of otherpur regulon genes by the purine repressor (PurR) (9, 15). PurR requires binding of hypoxanthine or guanine corepressors for interaction with a conserved 16-bp operator sequence (16,23). The position of the operator relative to the promoter varies among the pur regulon genes and in part determines the extent and mechanism of transcriptional regulation. In purB, the operator is far downstream from the promoter in the protein coding region (9). purB is regulated only about two-to threefold, whereas genes dedicated to the de novo synthesis of IMP are more highly regulated (8,9,15).Here, we report the results of experiments to determine the regulatory mechanism for purA. The purA gene is subject to dual regulation by levels of purines in the medium. One mechanism, described here, involves PurR-operator control. In addition, there is purR-independent regulation of purA. MATERIALS AND METHODSBacterial strains, plasmids, and media. The strains and plasmids used in this work are listed in Table 1. LB and 2 x YT media (18) were used as rich media. The minimal growth medium has been described previously (8).Plasmid construction. To clone the purA control region, a segment of purA from nucleotides -478 to + 103, numbered relative to the start of transcription (Fig. 1) was subcloned into the EcoRI-BamHI site of pUC118, resulting in plasmid pBH140. In a similar way, a 178-bp fragment from nucleotides -72 to +103 was constructed and was ligated into the Ec...

The Escherichia coli Transcriptional Regulator MarA Directly Represses Transcription of purA and hdeA

“…Other proteins also repress purA and hdeA. The purA promoter is repressed by PurR in the presence of purines (32), and the promoter of hdeA is repressed by HN-S (33). The hdeA promoter can be recognized by both 70 and RpoS (23), and MarA can activate both 70-and RpoS-dependent promoters (34).…”

Adenylosuccinate Syntheatase: Recent Developments

“…Transcription of purA is best characterized in E. coli, where the gene is a part of the purine regulon under control of the PurR master repressor protein (He and Zalkin, 1994).…”

Kinetic and Thermodynamic Studies of Purine Repressor Binding to Corepressor and Operator DNA

“…F was measured by scintillation counting. The association rate constants were determined by fitting k obs at different protein concentrations into Equation 4.…”

“…PurR recognizes a series of conserved and partially symmetric DNA sequences upon binding to a corepressor ligand, guanine or hypoxanthine (1,(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). In the presence of PurR-purine holorepressor, RNA polymerase transcription of enzymes from the associated promoter region is inhibited, and biosynthesis of purine nucleotides correspondingly diminishes (1,(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20).…”