CRP-cyclic AMP dependent inhibition of the xylene-responsive σ(54)-promoter Pu in Escherichia coli.
The expression of σ(54)-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.
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, China.
Yuan-Tao
YT
Zhang
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, China ; Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Feng
F
Jiang
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, China.
Zhe-Xian
ZX
Tian
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, China.
Yi-Xin
YX
Huo
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, China ; Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Yi-Cheng
YC
Sun
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, China.
Yi-Ping
YP
Wang
D000242:Cyclic AMP / Q000494:pharmacology*; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism*; D018983:DNA Footprinting; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D016958:Pseudomonas putida / Q000201:enzymology; D051877:RNA Polymerase Sigma 54 / Q000235:genetics*; D014158:Transcription, Genetic; D014992:Xylenes / Q000494:pharmacology*
D002373:Cyclic AMP Receptor Protein; D014992:Xylenes; D000242:Cyclic AMP; D051877:RNA Polymerase Sigma 54