Determinants of interaction specificity of the Bacillus subtilis GlcT antitermination protein: functionality and phosphorylation specificity depend on the arrangement of the regulatory domains.
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.
Department of NMR-based Structural Biology, Max Planck Institute for iophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Sebastian
S
Himmel
Christopher P
CP
Zschiedrich
Stefan
S
Becker
He-Hsuan
HH
Hsiao
Sebastian
S
Wolff
Christine
C
Diethmaier
Henning
H
Urlaub
Donghan
D
Lee
Christian
C
Griesinger
Jörg
J
Stülke
D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000096:biosynthesis* / Q000235:genetics; D010766:Phosphorylation / Q000502:physiology; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; C115000:GlcT protein, Bacillus subtilis; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C031629:phosphoenolpyruvate-glucose phosphotransferase