Krishna C. Suddala

The HIV-1 TAR RNA plays key roles in viral genome architecture, transcription and replication. Previous structural analyses focused on its upper stem loop, which has served as a paradigm to study RNA structural dynamics. However, an imperfectly paired lower stem immediately abuts and stacks with the upper half, both of which are required for efficient HIV replication. Here, we report crystal structures of the full-length HIV-1 TAR which reveal substantial conformational mobility in its three conserved bulges and in its lower stem, which coordinately maintain the structural fluidity of the entire RNA. We find that TAR RNA is a robust inhibitor of PKR, and primarily uses its lower stem to capture and sequester PKR monomers, preventing their dimerization and activation. The lower stem exhibits transient conformational excursions detected by a ligation assay. Time-resolved fluorescence spectroscopy reveals local and global TAR structural remodeling by HIV-1 nucleocapsid, Tat, and PKR. This study portrays the structure, dynamics, and interactions of a complete TAR RNA, uncovers a convergent RNA-based viral strategy to evade innate immunity, and provides avenues to develop antivirals that target a dynamic, multifunctional viral RNA.

RNA-Puzzles is a collective endeavor dedicated to the advancement and improvement of RNA three-dimensional structure prediction. With agreement from structural biologists, RNA structures are predicted by modeling groups before publication of the experimental structures. We report a large-scale set of predictions by 18 groups for 23 RNA-Puzzles: 4 RNA elements, 2 Aptamers, 4 Viral elements, 5 Ribozymes and 8 Riboswitches. We describe automatic assessment protocols for comparisons between prediction and experiment. Our analyses reveal some critical steps to be overcome to achieve good accuracy in modeling RNA structures: identification of helix-forming pairs and of non-Watson–Crick modules, correct coaxial stacking between helices and avoidance of entanglements. Three of the top four modeling groups in this round also ranked among the top four in the CASP15 contest.

T-box riboswitches are multi-domain noncoding RNAs that surveil individual amino acid availabilities in most Gram-positive bacteria. T-boxes directly bind specific tRNAs, query their aminoacylation status to detect starvation, and feedback control the transcription or translation of downstream amino-acid metabolic genes. Most T-boxes rapidly recruit their cognate tRNA ligands through an intricate three-way stem I-stem II-tRNA interaction, whose establishment is not understood. Using single-molecule FRET, SAXS, and time-resolved fluorescence, we find that the free T-box RNA assumes a broad distribution of open, semi-open, and closed conformations that only slowly interconvert. tRNA directly binds all three conformers with distinct kinetics, triggers nearly instantaneous collapses of the open conformations, and returns the T-box RNA to their pre-binding conformations upon dissociation. This scissors-like dynamic behavior is enabled by a hinge-like pseudoknot domain which poises the T-box for rapid tRNA-induced domain closure. This study reveals tRNA-chaperoned folding of flexible, multi-domain mRNAs through a Venus flytrap-like mechanism.

T-box riboswitches are modular bacterial noncoding RNAs that sense and regulate amino acid availability through direct interactions with tRNAs. Between the 5′ anticodon-binding stem I domain and the 3′ amino acid sensing domains of most T-boxes lies the stem II domain of unknown structure and function. Here, we report a 2.8-Å cocrystal structure of the Nocardia farcinica ileS T-box in complex with its cognate tRNA^Ile. The structure reveals a perpendicularly arranged ultrashort stem I containing a K-turn and an elongated stem II bearing an S-turn. Both stems rest against a compact pseudoknot, dock via an extended ribose zipper and jointly create a binding groove specific to the anticodon of its cognate tRNA. Contrary to proposed distal contacts to the tRNA elbow region, stem II locally reinforces the codon-anticodon interactions between stem I and tRNA, achieving low-nanomolar affinity. This study illustrates how mRNA junctions can create specific binding sites for interacting RNAs of prescribed sequence and structure.

The widespread Mn²⁺-sensing yybP-ykoY riboswitch controls the expression of bacterial Mn²⁺ homeostasis genes. Here, we first determine the crystal structure of the ligand-bound yybP-ykoY riboswitch aptamer from Xanthomonas oryzae at 2.96 Å resolution, revealing two conformations with docked four-way junction (4WJ) and incompletely coordinated metal ions. In >100 µs of MD simulations, we observe that loss of divalents from the core triggers local structural perturbations in the adjacent docking interface, laying the foundation for signal transduction to the regulatory switch helix. Using single-molecule FRET, we unveil a previously unobserved extended 4WJ conformation that samples transient docked states in the presence of Mg²⁺. Only upon adding sub-millimolar Mn²⁺, however, can the 4WJ dock stably, a feature lost upon mutation of an adenosine contacting Mn²⁺ in the core. These observations illuminate how subtly differing ligand preferences of competing metal ions become amplified by the coupling of local with global RNA dynamics.

T‐box riboswitches are a widespread class of structured noncoding RNAs in Gram‐positive bacteria that regulate the expression of amino acid‐related genes. They form negative feedback loops to maintain steady supplies of aminoacyl‐transfer RNAs (tRNAs) to the translating ribosomes. T‐box riboswitches are located in the 5′ leader regions of mRNAs that they regulate and directly bind to their cognate tRNA ligands. T‐boxes further sense the aminoacylation state of the bound tRNAs and, based on this readout, regulate gene expression at the level of transcription or translation. T‐box riboswitches consist of two conserved domains—a 5′ Stem I domain that is involved in specific tRNA recognition and a 3′ antiterminator/antisequestrator (or discriminator) domain that senses the amino acid on the 3′ end of the bound tRNA. Interaction of the 3′ end of an uncharged but not charged tRNA with a thermodynamically weak discriminator domain stabilizes it to promote transcription readthrough or translation initiation. Recent biochemical, biophysical, and structural studies have provided high‐resolution insights into the mechanism of tRNA recognition by Stem I, several structural models of full‐length T‐box‐tRNA complexes, mechanism of amino acid sensing by the antiterminator domain, as well as kinetic details of tRNA binding to the T‐box riboswitches. In addition, translation‐regulating T‐box riboswitches have been recently characterized, which presented key differences from the canonical transcriptional T‐boxes. Here, we review the recent developments in understanding the T‐box riboswitch mechanism that have employed various complementary approaches. Further, the regulation of multiple essential genes by T‐boxes makes them very attractive drug targets to combat drug resistance. The recent progress in understanding the biochemical, structural, and dynamic aspects of the T‐box riboswitch mechanism will enable more precise and effective targeting with small molecules. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1167–1180, 2019

The widespread manganese-ion sensing yybP-ykoY riboswitch controls the expression of bacterial Mn ²⁺ homeostasis genes. Here, we first determine the crystal structure of the ligand-bound yybP-ykoY riboswitch from Xanthomonas oryzae at 2.85 Å resolution, revealing two conformations with docked four-way junction (4WJ) and incompletely coordinated metal ions. In >50 μs of MD simulations, we observe that loss of divalents from the core triggers local structural perturbations in the adjacent docking interface, laying the foundation for signal transduction to the regulatory switch helix. Using single-molecule FRET, we unveil a previously unobserved extended 4WJ conformation that samples transient docked states in the presence of Mg ²⁺ . Only upon adding sub-millimolar Mn ²⁺ , however, can the 4WJ dock stably, a feature lost upon mutation of an adenosine contacting Mn ²⁺ in the core. These observations illuminate how subtly differing ligand preferences of competing metal ions become amplified by the coupling of local with global RNA dynamics.

Late endosome-resident interferon-induced transmembrane protein 3 (IFITM3) inhibits fusion of diverse viruses, including Influenza A virus (IAV), by a poorly understood mechanism. Despite the broad antiviral activity of IFITM3, viruses like Lassa virus (LASV), are fully resistant to its inhibitory effects. It is currently unclear whether resistance arises from a highly efficient fusion machinery that is capable of overcoming IFITM3 restriction or the ability to enter from cellular sites devoid of this factor. Here, we constructed and validated a functional IFITM3 tagged with EGFP or other fluorescent proteins. This breakthrough allowed live cell imaging of virus co-trafficking and fusion with endosomal compartments in cells expressing fluorescent IFITM3. Three-color single virus and endosome tracking revealed that sensitive (IAV), but not resistant (LASV), viruses become trapped within IFITM3-positive endosomes where they underwent hemifusion but failed to release their content into the cytoplasm. IAV fusion with IFITM3-containing compartments could be rescued by amphotericin B treatment, which has been previously shown to antagonize the antiviral activity of this protein. By comparison, virtually all LASV particles trafficked and fused with endosomes lacking detectable levels of fluorescent IFITM3, implying that this virus escapes restriction by utilizing endocytic pathways that are distinct from the IAV entry pathways. The importance of virus uptake and transport pathways is further reinforced by the observation that LASV glycoprotein-mediated cell-cell fusion is inhibited by IFITM3 and other members of the IFITM family expressed in target cells. Together, our results strongly support a model according to which IFITM3 accumulation at the sites of virus fusion is a prerequisite for its antiviral activity and that this protein traps viral fusion at a hemifusion stage by preventing the formation of fusion pores. We conclude that the ability to utilize alternative endocytic pathways for entry confers IFITM3-resistance to otherwise sensitive viruses. Expression of interferon-induced transmembrane proteins (IFITMs) in target cells potently inhibits fusion of many unrelated enveloped viruses, including the Influenza A virus, whereas arenaviruses, such as the Lassa fever virus, are resistant to these factors. The mechanism by which IFITMs interfere with the viral fusion step and the mechanism of virus escape from these restriction factors are poorly understood. Here, we tagged the late endosome-resident IFITM3 with fluorescent proteins and visualized single virus entry and fusion with endosomes in living cells expressing these constructs. Single virus and endosome tracking experiments demonstrate that the sensitive Influenza A virus is trapped within acidic IFITM3-positive endosomes that are not permissive for viral fusion. In contrast, the resistant Lassa virus consistently enters and fuses with endosomes lacking IFITM3. Our results imply that accumulation of IFITM3 in virus-carrying endosomes is a prerequisite for blocking fusion of diverse enveloped viruses and that viruses insensitive to this protein escape restriction by entering through distinct endosomal trafficking pathways that do not converge with IFITM3-positive compartments.

In Gram-positive bacteria, T-box riboswitches control gene expression to maintain the cellular pools of aminoacylated tRNAs essential for protein biosynthesis. Co-transcriptional binding of an uncharged tRNA to the riboswitch stabilizes an antiterminator, allowing transcription read-through, whereas an aminoacylated tRNA does not. Recent structural studies have resolved two contact points between tRNA and Stem-I in the 5′ half of the T-box riboswitch, but little is known about the mechanism empowering transcriptional control by a small, distal aminoacyl modification. Using single-molecule fluorescence microscopy, we have probed the kinetic and structural underpinnings of tRNA binding to a glycyl T-box riboswitch. We observe a two-step mechanism where fast, dynamic recruitment of tRNA by Stem-I is followed by ultra-stable anchoring by the downstream antiterminator, but only without aminoacylation. Our results support a hierarchical sensing mechanism wherein dynamic global binding of the tRNA body is followed by localized readout of its aminoacylation status by snap-lock-based trapping.

Bacterial riboswitches couple small-molecule ligand binding to RNA conformational changes that widely regulate gene expression, rendering them potential targets for antibiotic intervention. Despite structural insights, the ligand-mediated folding mechanisms of riboswitches are still poorly understood. Using single-molecule fluorescence resonance energy transfer (smFRET), we have investigated the folding mechanism of an H-type pseudoknotted preQ₁ riboswitch in dependence of Mg²⁺ and three ligands of distinct affinities. We show that, in the absence of Mg²⁺, both weakly and strongly bound ligands promote pseudoknot docking through an induced-fit mechanism. By contrast, addition of as low as 10 μM Mg²⁺ generally shifts docking toward conformational selection by stabilizing a folded-like conformation prior to ligand binding. Supporting evidence from transition-state analysis further highlights the particular importance of stacking interactions during induced-fit and of specific hydrogen bonds during conformational selection. Our mechanistic dissection provides unprecedented insights into the intricate synergy between ligand- and Mg²⁺-mediated RNA folding.