Roles of catabolite activator protein sites centered at -81.5 and -41.5 in the activation of the Klebsiella aerogenes histidine utilization operon hutUH

Abstract

The Klebsiella aerogenes hutUH operon is preceded by a promoter region, hut(P), that contains two divergent promoters (hutUp and Pc) which overlap and are alternately expressed. In the absence of the catabolite gene activator protein-cyclic AMP (CAP-cAMP) complex, Pc is predominantly expressed while hutUp is largely repressed. CAP-cAMP has the dual effect of repressing transcription from Pc while simultaneously activating transcription from hutUp. DNA deletion mutations in this region were used to identify DNA sequences required for transcription of these two promoters. We showed that inactivation of Pc by DNA deletion did not result in activation of hutUp in vitro or in vivo. In addition, Escherichia coli CAP mutants that are known to bind and bend DNA normally but are unable to activate various CAP-dependent promoters were also unable to activate hutUp in vivo. These results invalidate an indirect activation model by which CAP-mediated repression of Pc in itself would led to activation of hutUp. Gel retardation asays with various deletion mutations of hut(P) and DNase I protection analyses revealed a high-affinity CAP binding site (CAP site 1) centered at -81.5 relative to the hutUp start of transcription and a second low-affinity CAP site (CAP site 2) centered at about -41.5. CAP site 1 is essential for activation of hutUp. Although CAP site 2 by itself is unable to activate hutUp in vivo under catabolite-activating conditions, it appears to be required for maximal transcription from a site centered at -41.5, does not activate hutUp suggests that the role of CAP-cAMP at the weaker CAP site may be different from that of other promoters containing a similarly positioned site. We propose that CAP directly stimulates the activity of RNA polymerase at hutUp and that this reaction is completely dependent on a naturally occurring CAP site centered at -81.5 and also involves a second CAP site centered at about -41.5 for maximal activation.