Coupling Green Fluorescent Protein Expression with Chemical Modification to Probe Functionally Relevant Riboswitch Conformations in Live Bacteria

Noncoding RNAs engage in numerous biological activities including gene regulation. To fully understand RNA function it is necessary to probe biologically relevant conformations in living cells. To address this challenge, we coupled RNA-mediated regulation of the green fluorescent protein (GFP)­uv-reporter gene to icSHAPE (<u>i</u>n <u>c</u>ell <u>S</u>elective 2′-<u>H</u>ydroxyl <u>A</u>cylation analyzed by <u>P</u>rimer <u>E</u>xtension). Our transcript-specific approach provides sensitive, fluorescence-based readout of the regulatory-RNA status as a means to coordinate chemical modification experiments. We chose a plasmid-based reporter compatible with Escherichia coli to allow use of knockout strains that eliminate endogenous effector biosynthesis. The approach was piloted using the Lactobacillus rhamnosus (<i>Lrh</i>) preQ₁-II riboswitch, which senses the pyrrolopyrimidine metabolite preQ₁. Using an <i>E. coli</i> Δ<i>queF</i> strain incapable of preQ₁ anabolism, the <i>Lrh</i> riboswitch yielded nearly one log unit of GFPuv-gene repression resulting from exogenously added preQ₁. We then subjected cells in gene “on” and “off” states to icSHAPE. The resulting differential analysis indicated reduction in <i>Lrh</i> riboswitch flexibility in the P3 helix of the pseudoknot, which comprises the ribosome-binding site (RBS) paired with the anti-RBS. Such expression platform modulation was not observed by <i>in vitro</i> chemical probing and demonstrates that the crowded cellular environment does not preclude detection of compact and loose RNA-regulatory conformations. Here we describe the design, methods, interpretation, and caveats of <u>Re</u>porter <u>Co</u>upled (ReCo) icSHAPE. We also describe mapping of the differential ReCo-icSHAPE results onto the <i>Lrh</i> riboswitch-preQ₁ cocrystal structure. The approach should be readily applicable to functional RNAs triggered by effectors or environmental variations

Core-Binding Factor β Increases the Affinity between Human Cullin 5 and HIV‑1 Vif within an E3 Ligase Complex

HIV-1 Vif masquerades as a receptor for a cellular E3 ligase harboring Elongin B, Elongin C, and Cullin 5 (EloB/C/Cul5) proteins that facilitate degradation of the antiretroviral factor APOBEC3G (A3G). This Vif-mediated activity requires human core-binding factor β (CBFβ) in contrast to cellular substrate receptors. We observed calorimetrically that Cul5 binds tighter to full-length Vif^(1–192)/EloB/C/CBFβ (<i>K</i>_d = 5 ± 2 nM) than to Vif^(95–192)/EloB/C (<i>K</i>_d = 327 ± 40 nM), which cannot bind CBFβ. A comparison of heat capacity changes supports a model in which CBFβ prestabilizes Vif^(1–192) relative to Vif^(95–192), consistent with a stronger interaction of Cul5 with Vif’s C-terminal Zn²⁺-binding motif. An additional interface between Cul5 and an N-terminal region of Vif appears to be plausible, which has therapeutic design implications

Structural analysis of a class III preQ1 riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics.

PreQ1-III riboswitches are newly identified RNA elements that control bacterial genes in response to preQ1 (7-aminomethyl-7-deazaguanine), a precursor to the essential hypermodified tRNA base queuosine. Although numerous riboswitches fold as H-type or HLout-type pseudoknots that integrate ligand-binding and regulatory sequences within a single folded domain, the preQ1-III riboswitch aptamer forms a HLout-type pseudoknot that does not appear to incorporate its ribosome-binding site (RBS). To understand how this unusual organization confers function, we determined the crystal structure of the class III preQ1 riboswitch from Faecalibacterium prausnitzii at 2.75 Å resolution. PreQ1 binds tightly (KD,app 6.5 ± 0.5 nM) between helices P1 and P2 of a three-way helical junction wherein the third helix, P4, projects orthogonally from the ligand-binding pocket, exposing its stem-loop to base pair with the 3' RBS. Biochemical analysis, computational modeling, and single-molecule FRET imaging demonstrated that preQ1 enhances P4 reorientation toward P1-P2, promoting a partially nested, H-type pseudoknot in which the RBS undergoes rapid docking (kdock ∼ 0.6 s(-1)) and undocking (kundock ∼ 1.1 s(-1)). Discovery of such dynamic conformational switching provides insight into how a riboswitch with bipartite architecture uses dynamics to modulate expression platform accessibility, thus expanding the known repertoire of gene control strategies used by regulatory RNAs

Structural analysis of a class III preQ1 riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics.

PreQ1-III riboswitches are newly identified RNA elements that control bacterial genes in response to preQ1 (7-aminomethyl-7-deazaguanine), a precursor to the essential hypermodified tRNA base queuosine. Although numerous riboswitches fold as H-type or HLout-type pseudoknots that integrate ligand-binding and regulatory sequences within a single folded domain, the preQ1-III riboswitch aptamer forms a HLout-type pseudoknot that does not appear to incorporate its ribosome-binding site (RBS). To understand how this unusual organization confers function, we determined the crystal structure of the class III preQ1 riboswitch from Faecalibacterium prausnitzii at 2.75 Å resolution. PreQ1 binds tightly (KD,app 6.5 ± 0.5 nM) between helices P1 and P2 of a three-way helical junction wherein the third helix, P4, projects orthogonally from the ligand-binding pocket, exposing its stem-loop to base pair with the 3' RBS. Biochemical analysis, computational modeling, and single-molecule FRET imaging demonstrated that preQ1 enhances P4 reorientation toward P1-P2, promoting a partially nested, H-type pseudoknot in which the RBS undergoes rapid docking (kdock ∼ 0.6 s(-1)) and undocking (kundock ∼ 1.1 s(-1)). Discovery of such dynamic conformational switching provides insight into how a riboswitch with bipartite architecture uses dynamics to modulate expression platform accessibility, thus expanding the known repertoire of gene control strategies used by regulatory RNAs

Structure of HIV TAR in complex with a Lab-Evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription

Natural and lab-evolved proteins often recognize their RNA partners with exquisite affinity. Structural analysis of such complexes can offer valuable insight into sequence-selective recognition that can be exploited to alter biological function. Here, we describe the structure of a lab-evolved RNA recognition motif (RRM) bound to the HIV-1 trans-activation response (TAR) RNA element at 1.80 Å-resolution. The complex reveals a trio of arginines in an evolved β2-β3 loop penetrating deeply into the major groove to read conserved guanines while simultaneously forming cation-π and salt-bridge contacts. The observation that the evolved RRM engages TAR within a double-stranded stem is atypical compared to most RRMs. Mutagenesis, thermodynamic analysis and molecular dynamics validate the atypical binding mode and quantify molecular contributions that support the exceptionally tight binding of the TAR-protein complex (K[subscript D,App] of 2.5 ± 0.1 nM). These findings led to the hypothesis that the β2-β3 loop can function as a standalone TAR-recognition module. Indeed, short constrained peptides comprising the β2-β3 loop still bind TAR (K[subscript D,App] of 1.8 ± 0.5 μM) and significantly weaken TAR-dependent transcription. Our results provide a detailed understanding of TAR molecular recognition and reveal that a lab-evolved protein can be reduced to a minimal RNA-binding peptide