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- Gly184 of the Escherichia coli cAMP receptor protein provides optimal context for both DNA binding and RNA polymerase interaction
- Matt N. Hicks , Sanjiva Gunasekara , Jose Serate , Jin Park , Pegah Mosharaf , Yue Zhou , Jin-Won Lee , Hwan Youn
- J. Microbiol. 2017;55(10):816-822. Published online September 28, 2017
- DOI: https://doi.org/10.1007/s12275-017-7266-x
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Abstract
- The Escherichia coli cAMP receptor protein (CRP) utilizes the helix-turn-helix motif for DNA binding. The CRP’s recognition helix, termed F-helix, includes a stretch of six amino acids (Arg180, Glu181, Thr182, Val183, Gly184, and Arg185) for direct DNA contacts. Arg180, Glu181 and Arg185 are known as important residues for DNA binding and specificity, but little has been studied for the other residues. Here we show that Gly184 is another F-helix residue critical for the transcriptional activation function of CRP. First, glycine was repeatedly selected at CRP position 184 for its unique ability to provide wild type-level transcriptional activation activity. To dissect the glycine requirement, wild type CRP and mutants G184A, G184F, G184S, and G184Y were purified and their in vitro DNA-binding activity was measured. G184A and G184F displayed reduced DNA binding, which may explain their low transcriptional activation activity. However, G184S and G184Y displayed apparently normal DNA affinity. Therefore, an additional factor is needed to account for the diminished transcriptional activation function in G184S and G184Y, and the best explanation is perturbations in their interaction with RNA polymerase. The fact that glycine is the smallest amino acid could not fully warrant its suitability, as shown in this study. We hypothesize that Gly184 fulfills the dual functions of DNA binding and RNA polymerase interaction by conferring conformational flexibility to the F-helix.
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cAMP-independent DNA binding of the CRP family protein DdrI from
Deinococcus radiodurans
Yudong Wang, Jing Hu, Xufan Gao, Yuchen Cao, Shumai Ye, Cheng Chen, Liangyan Wang, Hong Xu, Miao Guo, Dong Zhang, Ruhong Zhou, Yuejin Hua, Ye Zhao, Paul Babitzke
mBio.2024;[Epub] CrossRef - Unexpected Requirement of Small Amino Acids at Position 183 for DNA Binding in the Escherichia coli cAMP Receptor Protein
Marcus Carranza, Amanda Rea, Daisy Pacheco, Christian Montiel, Jin Park, Hwan Youn
Journal of Microbiology.2024; 62(10): 871. CrossRef - cAMP Activation of the cAMP Receptor Protein, a Model Bacterial Transcription Factor
Hwan Youn, Marcus Carranza
Journal of Microbiology.2023; 61(3): 277. CrossRef
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cAMP-independent DNA binding of the CRP family protein DdrI from
Deinococcus radiodurans
- Mechanism of Transcriptional Activation of the Phosphate Regulon in Escherichi coli
- Kozo Makino , Mitsuko Amemura , Soo-Ki Kim , Katsushi Yokoyama , Sigenobu Kimura
- J. Microbiol. 1998;36(4):231-238.
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Abstract
- In Escherichia coli, at least 31 genes, which are involved in the roles related to the transport and assimilation of phosphate and phosphorus compounds, are induced by phosphate starvation. They constitute a single phosphate (pho) regulon, and are under the same physiological and genetic control (30, 36, 46). Proteins PhoB and PhoR, which are regulatory systems for the transcriptional regulation of the phogenes, belong to a large family of two-component regulatory systems that respond to a variety of environmental stimuli in bacteria (23, 24, 33, 39). PhoB is the transcriptional activator, which binds to the promoters of the pho genes (21, 22). PhoR is a transmembrane protein that modulates the activity of PhoB by promoting specific phosphorylation and dephosphorylation of PhoB in response to the phosphate signal in the medium (19, 21, 37, 50). The phosphorylation of PhoB protein occurs concurrently with the acquisition of the ability to activate transcription from the pho promoters (Fig. 1). In the absence of the PhoR functions, PhoB is phosphorylated independently of the phosphate levels by PhoM, a PhoR like protein (2, 3, 26), which was renamed CreC by Wanner (45). In this article, we describe our recent studies on the mechanism of the transcriptional regulation of the pho regulon.
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