A decreased intracellular concentration of cAMP is insufficient to account for catabolite repression in Escherichia coli. We show that glucose lowers the amount of cAMP receptor protein (CRP) in cells. A correlation exists between CRP and beta-galactosidase levels in cells growing under various conditions. Exogenous cAMP completely eliminates catabolite repression in CRP-overproducing cells, while it does not fully reverse the effect of glucose on beta-galactosidase expression in wild-type cells. When the CRP concentration is reduced by manipulating the crp gene, beta-galactosidase expression decreases in proportion to the concentration of CRP. These findings indicate that the lowered concentration of CRP caused by glucose is one of the major factors for catabolite repression. We propose that glucose causes catabolite repression by lowering the intracellular levels of both CRP and cAMP.

Glucose causes catabolite repression by lowering the intracellular levels of both cAMP and cAMP receptor protein (CRP) in Escherichia coli. The molecular mechanism underlying the down‐regulation of CRP by glucose has been investigated. We show that glucose lowers the level of crp mRNA without affecting its stability. Replacement of the crp promoter with the bla promoter almost completely abolishes the glucose‐mediated regulation of crp expression. Only a slight reduction in the crp expression by glucose is observed in cya‐ or crp‐ strains, suggesting that a CRP‐cAMP complex is needed for this regulation. We previously showed that transcription of the crp gene is regulated both negatively and positively. Positive autoregulation of crp is caused by the binding of CRP‐cAMP to the CRP binding site II located upstream of the crp promoter. Here we show that disrupting the CRP binding site II essentially eliminates the down‐regulation of crp expression by glucose. We conclude that the autoregulatory circuit of the crp gene plays a key role in the down‐regulation of CRP by glucose.

The role of the a subunit of Escherichia coli RNA polymerase in transcription activation by positive factors was investigated using two reconstituted mutant RNA polymerases (containing C-terminally truncated a subunits) and three positive factors [the cAMP receptor protein (CRP), OmpR, and PhoB]. The mutant RNA polymerases did not respond to transcription activation by activator proteins that bind upstream of the respective promoters. Transcription by these mutant enzymes was, however, activated in the cases where activators bind to target sites that overlap the promoter -35 region. Two different mechanisms are proposed for the positive control of transcription by activator proteins, one requiring the C-terminal domain of the a subunit, and the other not requiring it.Positive control of transcription is one of the common mechanisms of regulated gene expression in both prokaryotes and eukaryotes. Two mechanisms have been proposed for this activation, (i) lacking the C-terminal 73 or 94 amino acid residues (9). The mutant RNA polymerase core enzymes containing C-terminally truncated a subunits exhibited essentially the same specific activity of RNA synthesis as the native enzyme. After addition of o70 subunit, these mutant holoenzymes were able to initiate transcription accurately from certain promoters. However, they did not respond to transcription activation by cAMP/CRP at two type I CRP-dependent promoters on the lac and uxuAB genes (9). These results led us to conclude that the C-terminal domain (residues 257-329) ofthe a subunit is involved in the response of RNA polymerase to transcriptional activation by cAMP/CRP. Phenotypes resulting from known rpoA mutations also suggest that the a subunit is involved in positive control by certain transcription activators (10-14).In the present study, we extended this line of experiments to type II CRP-dependent promoters. Most CRP-dependent promoters can be classified into two types depending on the location of the CRP site relative to the transcription start site (15, 16). Type I promoters are associated with a CRP site that is separated from the basic promoter (-10 and -35 signals) and include the lac P1 and uxuAB promoters. On type II promoters, the CRP site is centered around -41/-42 and therefore partially overlaps the basic promoter at the -35 region. In addition, we examined the responsiveness of the mutant RNA polymerases to transcription stimulation by two other activators, PhoB, an activator of the phosphate regulon, and OmpR, which is involved in osmoregulation of the ompF and ompC genes (reviewed in ref. 17). The results suggest the presence oftwo different mechanisms for positive control of transcription by activator proteins: one requires the C-terminal domain of the a subunit, and the other does not. MATERIALS AND METHODSRNA Polymerases. Wild-type and mutant RNA polymerases were assembled in vitro from individually overproduced and purified (3, (3', and (770 subunits and one of the wild-type or mutant a subunits, as described by Igarashi and...

Transcription of the Escherichia coli crp gene encoding cAMP receptor protein (CRP) is negatively regulated by CRP-cAMP complex that binds to a specific site located downstream from the transcription start site. The binding of CRP-cAMP to this site activates transcription from a second divergent overlapping promoter. The mechanism of this negative autoregulation of the crp gene has been investigated by in vitro transcription, gel shift, DNase I footprinting, and exonuclease III protection assays. We demonstrated that the crp and divergent promoters are reciprocally and coordinately regulated by CRP-cAMP. The abortive initiation assay revealed that the divergent RNA itself is not required for the inhibition of crp transcription. Detailed binding studies revealed that CRP-cAMP stimulates the binding of RNA polymerase to the divergent promoter and thus blocks the occupation of the crp promoter by RNA polymerase.