Abstract
LicT belongs to a family of bacterial transcriptional antiterminators, which control the expression of sugar-metabolizing operons in response to phosphorylations by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Previous studies of LicT have revealed the structural basis of RNA recognition by the dimeric N-terminal co-antiterminator (CAT) domain on the one hand and the conformational changes undergone by the duplicated regulation domain (PRD1 and PRD2) upon activation on the other hand. To investigate the mechanism of signal transduction between the effector and regulation modules, we have undertaken the characterization of a fragment, including the CAT and PRD1 domains and the linker in-between. Comparative experiments, including RNA binding assays, NMR spectroscopy, limited proteolysis, analytical ultracentrifugation, and circular dichroism, were conducted on native CAT-PRD1 and on a constitutively active CAT-PRD1 mutant carrying a D99N substitution in PRD1. We show that in the native state, CAT-PRD1 behaves as a rather unstable RNA-binding deficient dimer, in which the CAT dimer interface is significantly altered and the linker region is folded as a trypsin-resistant helix. In the activated mutant form, the CAT-PRD1 linker becomes protease-sensitive, and the helix content decreases, and the CAT module adopts the same dimeric conformation as in isolated CAT, thereby restoring the affinity for RNA. From these results, we propose that a helix-to-coil transition in the linker acts as the structural relay triggered by the regulatory domain for remodeling the effector dimer interface. In essence, the structural mechanism modulating the LicT RNA antitermination activity is thus similar to that controlling the DNA binding activity of dimeric transcriptional regulators.
Highlights
For over 2 decades, we have been studying transcriptional regulation by a family of antitermination proteins controlling the hierarchical utilization of carbohydrates in bacteria
Our previous modular studies of LicT revealed the structural basis of RNA recognition by the isolated N-terminal CAT domain on one hand, as well as the conformational changes undergone by the tandem phosphotransferase system (PTS) regulation domains upon activation on the other hand [15, 18, 21, 41]
We showed that adding the first PTS regulation module (PRD1) to the CAT domain led to a loss of RNA binding activity in vitro
Summary
II (BglP) inhibits antitermination, whereas phosphorylation of the PRD2 histidines by the general histidine-phosphocarrier protein leads to LicT activation and transcription of the bgl genes [12,13,14]. In the activated mutant structure, both PRD1 and PRD2 form tight dimers, whereas in the native construct most of the dimeric interface is provided by PRD1-PRD1 contacts, and PRD2 becomes almost monomeric (see Fig. 1b) These studies clearly brought to light massive activation-dependent structural changes in the LicT regulatory region, they did not reveal how this regulation signal is transmitted from the sensor domain to the effector domain. The conformational differences between the wild type and D99N mutant CAT-PRD1 were investigated by NMR spectroscopy, analytical ultracentrifugation, circular dichroism, and limited proteolysis experiments The results of these analyses revealed that important rearrangements of the CAT dimeric interface are driven by PRD1, not through a direct contact between the two domains but through the helical linker, which appears to unfold upon activation. Our data provide the first structural insights into the mechanism of signal transduction between the regulatory and RNAbinding domains within an antitermination protein of the BglG/SacY family
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
