Competitive folding of RNA structures at a termination–antitermination site

Abstract

Antitermination is a regulatory process based on the competitive folding of terminator-antiterminator structures that can form in the leader region of nascent transcripts. In the case of the gene involved in β-glucosides utilization, the binding of the antitermination protein LicT to a short RNA hairpin (RAT) prevents the formation of an overlapping terminator and thereby allows transcription to proceed. Here, we monitored in vitro the competition between termination and antitermination by combining bulk and single-molecule fluorescence-based assays using labeled RNA oligonucleotide constructs of increasing length that mimic the progressive transcription of the terminator invading the antiterminator hairpin. Although high affinity binding is abolished as soon as the antiterminator basal stem is disrupted by the invading terminator, LicT can still bind and promote closing of the partially unfolded RAT hairpin. However, binding no longer occurs once the antiterminator structure has been disrupted by the full-length terminator. Based on these findings, we propose a kinetic competition model for the sequential events taking place at the termination-antitermination site, where LicT needs to capture its RAT target before completion of the terminator to remain tightly bound during RNAP pausing, before finally dissociating irreversibly from the elongated transcript.

Structural Insights into of the Allosteric Activation of the LicT Antiterminator by PTS-Mediated Phosphorylation

“…Fluorescence polarization was measured at 25 C using a Tecan Saphire II microplate reader, with the excitation set at 630 nM and the emission at 680 nm +/-10 nm. Affinity constants were estimated by fitting the titration curves using either a two-site or one-site affinity model (Ait-Bara et al, 2017).…”

“…In its activated state, LicT binds to nascent mRNA at a short palindromic ribonucleic antiterminator (RAT) sequence overlapping a regulatory transcriptional terminator upstream of bglP and licS (Tortosa et al, 2001). LicT binding stabilizes the stem-loop structure adopted by the RAT and prevents the formation of a competing terminator hairpin that otherwise promotes transcription arrest (Ait-Bara et al, 2017;Clerte et al, 2013).…”

“…Activation of LicT by PTS-Mediated Phosphorylation(A) Fluorescence anisotropy titrations of Atto647N-labeled licS-RAT RNA (0.5 nM, sequence in FigureS4A) by native LicT or the activated mutant LicT* after incubation in the phosphorylation buffer in the absence (dotted lines) or presence (plain lines) of the purified PTS general components EI and HPr. Data were fitted using a simple binding model, or when appropriate, a double binding model of high (nM) and low (mM) affinity as described previously(Ait-Bara et al, 2017). (B) SEC elution profiles of native LicT and activated LicT* before (LicT and LicT*) and phosphorylation (LicT-P and LicT*-P).…”

Ultraviolet Nanophotonics Enables Autofluorescence Correlation Spectroscopy on Label-Free Proteins with a Single Tryptophan

“…TNase was selected because its single Trp residue was theoretically predicted to have a quantum yield of 28% in good agreement with ensemble spectroscopy measurements . TNase is also widely studied as a model system in protein chemistry. , LicT was selected for its UV signal being comparable to TNase and its availability in purified form labeled with Cy3B to serve as a control using visible fluorescence spectroscopy. , Streptavidin was selected because of its higher number of Trps, large availability, moderate mass, and good water solubility.…”

“…42,43 LicT was selected for its UV signal being comparable to TNase and its availability in purified form labeled with Cy3B to serve as a control using visible fluorescence spectroscopy. 44,45 Streptavidin was selected because of its higher number of Trps, large availability, moderate mass, and good water solubility. 20 The limits of detection for the confocal 18 and the optical horn antenna (this work) are indicated.…”