Structural Mechanism of Signal Transduction between the RNA-binding Domain and the Phosphotransferase System Regulation Domain of the LicT Antiterminator

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 inbetween. 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 RNAbinding deficient dimer, in which the CAT dimer interface is significantly altered and the linker region is folded as a trypsinresistant 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.

Competitive folding of anti-terminator/terminator hairpins monitored by single molecule FRET

“…As expected, the LicT activating mutations leads to an increase in binding affinity (from 72 ± 6 to 1.8 ± 0.3 nM), consistent with the qualitative increase in binding affinity observed using gel-shift assays or surface plasmon resonance (19), and with the 40-fold increase in transcription anti-termination efficiency in vivo reported for this constitutive mutant relative to the WT (17). The K d values measured here for the native and activated forms of full-length LicT are also in good agreement with the binding constants estimated previously for native and activated LicT CAT-PRD1 constructs in which the RNA-binding domain is fused to a WT or mutant PRD1 (15).

Figure 2.Fluorescence anisotropy titrations of anti-terminator hairpins by full-length LicT or LicT- and SacY-CAT domains.

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“…From a structural point of view, LicT is by far the best characterized member of the BglG family (14,15). The activation of LicT involves a massive conformational rearrangement of the relative orientation of the monomers, which in turn results in the partial unfolding of the CAT-PRD1 linker helix, allowing CAT domains to dimerize and interact efficiently with RNA (15,16).…”

“…The activation of LicT involves a massive conformational rearrangement of the relative orientation of the monomers, which in turn results in the partial unfolding of the CAT-PRD1 linker helix, allowing CAT domains to dimerize and interact efficiently with RNA (15,16). This activation is constitutive in the H207D/H269D double mutant (named LicT* herein), which mimics the phosphorylation of the corresponding histidines, as well as in isolated CAT domains (17).…”

Termination and antitermination: RNA polymerase runs a stop sign

“…The structural basis of the underlying molecular mechanisms has been described for only a few RNA-binding regulators. Among these are antiterminator proteins His utilization ( hut ) operon positive regulatory protein (HutP) 40 , PtsGHI operon antiterminator GlcT 41 and transcription antiterminator LicT 42 .…”

“…If glucose is present, the phosphate groups are transferred from PtsG to the sugar instead. In LicT, an antiterminator homologous to GlcT that controls transport and metabolism of β-glucosides, the sensor domain is connected to the RNA-binding domain by a linker that undergoes a helix–coil transition upon inducer binding to allow LicT to bind its RNA target 42 . The related antiterminator, BglG, is regulated both by reversible phosphorylation and by sequestration into an inactive complex 45 .…”