Downregulation of the Escherichia coli guaB Promoter by Upstream-Bound Cyclic AMP Receptor Protein

Abstract

The Escherichia coli guaB promoter (P guaB ) is responsible for directing transcription of the guaB and guaA genes, which specify the biosynthesis of the nucleotide GMP. P guaB is subject to growth rate-dependent control (GRDC) and possesses an UP element that is required for this regulation. In addition, P guaB contains a discriminator, three binding sites for the nucleoid-associated protein FIS, and putative binding sites for the regulatory proteins DnaA, PurR, and cyclic AMP receptor protein (CRP). Here we show that the CRP-cyclic AMP (cAMP) complex binds to a site located over 100 bp upstream of the guaB transcription start site, where it serves to downregulate P guaB . The CRP-mediated repression of P guaB activity increases in media that support lower growth rates. Inactivation of the crp or cyaA gene or ablation/translocation of the CRP site relieves repression by CRP and results in a loss of GRDC of P guaB . Thus, GRDC of P guaB involves a progressive increase in CRP-mediated repression of the promoter as the growth rate decreases. Our results also suggest that the CRP-cAMP complex does not direct GRDC at P guaB and that at least one other regulatory factor is required for conferring GRDC on this promoter. However, PurR and DnaA are not required for this regulatory mechanism.The Escherichia coli guaB promoter (P guaB ) regulates transcription of the guaBA operon. The guaB and guaA genes encode IMP dehydrogenase and GMP synthetase, respectively, and are required for synthesis de novo of GMP from the common purine precursor IMP (46, 75). P guaB responds to a variety of physiological signals. For example, the activity of P guaB increases as a function of the cellular growth rate, such that guaBA mRNA forms an increasing fraction of total cell mass at higher growth rates (16,33,34). This form of regulation is referred to as growth rate-dependent control (GRDC) (17, 27). P guaB is also subject to stringent control (16, 71), growth phase-dependent regulation (34), and purine repression (16,70), and its activity is coupled to the DNA replication cycle (73).The multivalent regulation of P guaB is reflected by the presence of a number of cis-acting regulatory sites that overlap this promoter. An UP element, located immediately upstream of the promoter Ϫ35 region, strongly enhances transcription and is required for GRDC of this promoter (28, 33). Three binding sites for the nucleoid-associated protein FIS have been identified, centered near positions Ϫ11, ϩ8, and ϩ29 relative to the guaB transcription start site. Accordingly, FIS has been shown to repress transcription from P guaB in vitro. However, FIS is not required for GRDC of P guaB (34). P guaB also contains a putative binding site for PurR that overlaps the core promoter region (16, 31) (see Fig. 1). Consistent with this, IMP dehydrogenase activity is higher in a purR mutant strain, and unlike the situation in the wild-type strain, this activity is not repressed by growth in the presence of high concentrations of guanine or guanosine (46,48). Approximately ...

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