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

PLOS ONE, Dec 2019

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.

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

et al. (2014) CRP-Cyclic AMP Dependent Inhibition of the Xylene-Responsive s54-Promoter Pu in Escherichia coli. PLoS ONE 9(1): e86727. doi:10.1371/journal.pone.0086727 CRP-Cyclic AMP Dependent Inhibition of the Xylene- 54 Responsive s -Promoter Pu in Escherichia coli Yuan-Tao Zhang 0 Feng Jiang 0 Zhe-Xian Tian 0 Yi-Xin Huo 0 Yi-Cheng Sun 0 Yi-Ping Wang 0 Dipankar Chatterji, Indian Institute of Science, India 0 1 State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University , Beijing , China , 2 Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College , Beijing , China The expression of s54-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 aCTD (C-terminal domain of a-subunit) of RNA polymerase, was found essential for the CRP-mediated inhibition at Pu promoter. A model for the above observations is discussed. - Funding: This work was supported by 973 National Key Basic Research Program in China [No. 2010CB126503, YPW is the chief scientist of the program]; the National Natural Science Foundation of China [No. 30830005 to YPW]; the State Key Laboratory of Protein and Plant Gene Research [No. B02]; YPW is recipient of the National Science Fund for Distinguished Young Scholars [NSFC, No. 39925017]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Current address: Chemical and Biomedical Engineering Department, University of California Los Angeles, Los Angeles, California, United States of America The s54-dependent Pu promoter drives transcription of the upper operon of Pseudomonas putida mt-2 TOL plasmid pWW0 for degradation of toluene and xylenes [14]. This promoter region includes two upstream activating sites (UASs) for the activator protein XylR [5,6], a 212/224 region recognized by Es54 RNA polymerase, a single integration host factor (IHF) binding site located in the intervening region[5,7] and the adjacent UP-like elements for docking of the Es54 [8]. IHF-mediated DNA bending facilitates the direct interactions between two CTDs of the RNA polymerase and two separated UP-like elements located 278 and 2104 upstream of the transcriptional start site [8], recruiting Es54 to the Pu promoter when cells reach stationary phase [9,10]. It is generally believed that this IHF-dependent closed complex formation is the rate-limiting step for the transcriptional initiation at the Pu promoter [8,11]. Recently, the architectural organization of the s54-dependent promoter was investigated and led to the conclusion that the activator must approach the Es54 closed complexes from the unbound (activator accessible) face of the promoter DNA helix to catalyze open complex formation [12]. This conclusion is further supported by the first modeling of activator-promoter DNA-Es54 complex [12]. Since the contact between the UAS bound activator and promoter bound Es54 depends on the orientation of the DNA bending between UAS and 212/224 region of a promoter [13,14], the optimal IHF induced DNA bending at Pu promoter is essential for the transcription initiation [8]. CRP, the cyclic AMP (cAMP) receptor protein, is one of the best studied transcriptional factor, which is responsible for the regulation of more than 100 genes mainly involved in catabolism of sugars, amino acids and nucleotides in E.coli [15,16]. The CRPmediated regulation requires initially the binding of cAMP to form an active CRP homodimer when intracellular cAMP level is high, but in the presence of PTS (phosphoenolpyruvate-sugar phosphotransferase system)-sugars such as glucose the low cAMP level diminishes the activity of CRP [16]. At the s70-dependent promoters, the dimeric CRP protein enhances the ability of Es70 to bind DNA and initiate transcription by interacting with Es70 directly [17]. Two discrete surfaces of CRP, known as Activating Region 1 (AR1, consisting of residues 156164 of CRP) and Activating Region (...truncated)


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Yuan-Tao Zhang, Feng Jiang, Zhe-Xian Tian, Yi-Xin Huo, Yi-Cheng Sun, Yi-Ping Wang. CRP-Cyclic AMP Dependent Inhibition of the Xylene-Responsive σ54-Promoter Pu in Escherichia coli, PLOS ONE, 2014, Volume 9, Issue 1, DOI: 10.1371/journal.pone.0086727