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.
Highlights
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 [1,2,3,4]. 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]
XylR-mediated activation of the P. putida Pu promoter was recreated in the heterologous host E. coli
Plasmid pKU700 was co-transformed with pTS174 into E. coli cya mutant TP2006 and its isogenic wild type TP2101 separately. b-galactosidase activities were measured in the presence of different concentrations of m-methylbenzyl alcohol (mMBA), an effective aromatic inducer for XylR [32]
Summary
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 [1,2,3,4] 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]. 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]. 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]
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