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Journal Article

Determinants of iinteraction specificity of the Bacillus subtilis GlcT antitermination protein: Funktionality and phosphorylation specificity depend on the arrangement of the regulatory domains.

MPS-Authors
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Himmel,  S.
Department of NMR Based Structural Biology, MPI for biophysical chemistry, Max Planck Society;

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Becker,  S.
Department of NMR Based Structural Biology, MPI for biophysical chemistry, Max Planck Society;

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Hsiao,  H. H.
Research Group of Bioanalytical Mass Spectrometry, MPI for biophysical chemistry, Max Planck Society;

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Wolff,  S.
Department of NMR Based Structural Biology, MPI for biophysical chemistry, Max Planck Society;

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Urlaub,  H.
Research Group of Bioanalytical Mass Spectrometry, MPI for biophysical chemistry, Max Planck Society;

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Griesinger,  C.
Department of NMR Based Structural Biology, MPI for biophysical chemistry, Max Planck Society;

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Fulltext (public)

1546195.pdf
(Publisher version), 3MB

Supplementary Material (public)

1546195_1.pdf
(Supplementary material), 894KB

Citation

Himmel, S., Zschiedrich, C. P., Becker, S., Hsiao, H. H., Wolff, S., Dietmaier, C., et al. (2012). Determinants of iinteraction specificity of the Bacillus subtilis GlcT antitermination protein: Funktionality and phosphorylation specificity depend on the arrangement of the regulatory domains. Journal of Biological Chemistry, 287(33), 27731-27742. doi:10.1074/jbc.M112.388850.


Cite as: http://hdl.handle.net/11858/00-001M-0000-000F-F0F0-4
Abstract
The control of several catabolic operons in bacteria by transcription antitermination is mediated by RNA-binding proteins that consist of an RNA-binding domain and two reiterated phosphotransferase system regulation domains (PRDs). The Bacillus subtilis GlcT antitermination protein regulates the expression of the ptsG gene, encoding the glucose-specific enzyme II of the phosphotransferase system. In the absence of glucose, GlcT becomes inactivated by enzyme II-dependent phosphorylation at its PRD1, whereas the phosphotransferase HPr phosphorylates PRD2. However, here we demonstrate by NMR analysis and mass spectrometry that HPr also phosphorylates PRD1 in vitro but with low efficiency. Size exclusion chromatography revealed that non-phosphorylated PRD1 forms dimers that dissociate upon phosphorylation. The effect of HPr on PRD1 was also investigated in vivo. For this purpose, we used GlcT variants with altered domain arrangements or domain deletions. Our results demonstrate that HPr can target PRD1 when this domain is placed at the C terminus of the protein. In agreement with the in vitro data, HPr exerts a negative control on PRD1. This work provides the first insights into how specificity is achieved in a regulator that contains duplicated regulatory domains with distinct dimerization properties that are controlled by phosphorylation by different phosphate donors. Moreover, the results suggest that the domain arrangement of the PRD-containing antitermination proteins is under selective pressure to ensure the proper regulatory output, i.e. transcription antitermination of the target genes specifically in the presence of the corresponding sugar.