CRP-Cyclic AMP Dependent Inhibition of the Xylene-Responsive σ54-Promoter Pu in Escherichia coli

Abstract

The expression of σ54-dependent Pseudomonas putida Pu promoter is activated by XylR activator when cells are exposed to a variety of aromatic inducers. In this study, the transcriptional activation of the P. putida Pu promoter was recreated in the heterologous host Escherichia coli. Here we show that the cAMP receptor protein (CRP), a well-known carbon utilization regulator, had an inhibitory effect on the expression of Pu promoter in a cAMP-dependent manner. The inhibitory effect was not activator specific. In vivo KMnO4 and DMS footprinting analysis indicated that CRP-cAMP poised the RNA polymerase at Pu promoter, inhibiting the isomerization step of the transcription initiation even in the presence of an activator. Therefore, the presence of PTS-sugar, which eliminates cAMP, could activate the poised RNA polymerase at Pu promoter to transcribe. Moreover, the activation region 1 (AR1) of CRP, which interacts directly with the αCTD (C-terminal domain of α-subunit) of RNA polymerase, was found essential for the CRP-mediated inhibition at Pu promoter. A model for the above observations is discussed.

MrpC, a CRP/Fnr homolog, functions as a negative autoregulator during the Myxococcus xanthus multicellular developmental program

“…Similarly, direct interactions between E . coli CRP and the σ 54 ‐RNAP complex can either produce an alternative closed promoter complex (Wang et al ., ) or disrupt the conversion from closed promoter complex to an open promoter complex on the Pseudomonas putida Pu promoter (Zhang et al ., ). In a native system, E. coli CRP binding to the glnA p2 promoter induces DNA bending that prevents the bEBP NtrC from interacting with the σ 54 ‐RNAP complex (Huo et al ., ).…”

“…First, E. coli CRP has been shown to negatively regulate the expression of Sinorhizobium meliloti dctA by directly competing with its bEBP, DctD, for binding at the UAS sites (Wang et al, 1993(Wang et al, , 1998. Similarly, direct interactions between E. coli CRP and the r 54 -RNAP complex can either produce an alternative closed promoter complex (Wang et al, 1998) or disrupt the conversion from closed promoter complex to an open promoter complex on the Pseudomonas putida Pu promoter (Zhang et al, 2014). In a native system, E. coli CRP binding to the glnAp2 promoter induces DNA bending that prevents the bEBP NtrC from interacting with the r 54 -RNAP complex (Huo et al, 2006).…”

“…On UAS binding, activated bEBPs oligomerize, directly contact the r 54 -RNAP via DNA looping, and hydrolyze ATP to stimulate open complex formation [reviewed in (Bush and Dixon, 2012)]. cAMP-CRP has been shown to regulate r 54 -dependent promoters important for the metabolism of alternative carbon sources (Lee et al, 2005;Zhang et al, 2014), and nitrogen assimilation (Mao et al, 2007). Since activation of the bEBP, CRP and r 54 availability each depend on distinct stimuli, such systems function to integrate and coordinate information.…”