Group II intron ribozymes fold into their native structure by a unique stepwise process that involves an initial slow compaction followed by fast formation of the native state in a Mg 2+ -dependent manner. Single-molecule fluorescence reveals three distinct on-pathway conformations in dynamic equilibrium connected by relatively small activation barriers. From a most stable near-native state, the unobserved catalytically active conformer is reached. This most compact conformer occurs only transiently above 20 mM Mg 2+ and is stabilized by substrate binding, which together explain the slow cleavage of the ribozyme. Structural dynamics increase with increasing Mg 2+ concentrations, enabling the enzyme to reach its active state.

Spliceosomes catalyze the maturation of precursor mRNAs from yeast to humans. Their catalytic core comprises three small nuclear RNAs (U2, U5 and U6) involved in substrate positioning and catalysis. It has been postulated, but never shown experimentally, that the U2/U6 complex adopts at least two conformations that reflect different activation states. We have used single-molecule fluorescence to probe the structural dynamics of a protein-free RNA complex modeling U2/U6 from yeast and mutants of highly conserved regions. Our data show the presence of at least three distinct conformations in equilibrium. The minimal folding pathway consists of a two-step process with an obligatory intermediate. The first step is strongly magnesium dependent and we provide evidence suggesting the second corresponds to the formation of the genetically conserved helix IB. Site-specific mutations in the highly conserved AGC triad and the U80 base in U6 suggest that the observed conformational dynamics correlate with residues that play an important role in splicing.

Gut microbes play a key role in human health and nutrition by catabolizing a wide variety of glycans via enzymatic activities that are not encoded in the human genome. The ability to recognize and process carbohydrates strongly influences the structure of the gut microbial community. While the effects of diet on the microbiota are well documented, little is known about the molecular processes driving metabolism. To provide mechanistic insight into carbohydrate catabolism in gut symbionts, we studied starch processing in real time in the model Bacteroides thetaiotaomicron starch utilization system (Sus) by single-molecule fluorescence. Although previous studies have explored Sus protein structure and function, the transient interactions, assembly, and collaboration of these outer membrane proteins have not yet been elucidated in live cells. Our live-cell superresolution imaging reveals that the polymeric starch substrate dynamically recruits Sus proteins, serving as an external scaffold for bacterial membrane assembly of the Sus complex, which may promote efficient capturing and degradation of starch. Furthermore, by simultaneously localizing multiple Sus outer membrane proteins on the B. thetaiotaomicron cell surface, we have characterized the dynamics and stoichiometry of starch-induced Sus complex assembly on the molecular scale. Finally, based on Sus protein knockout strains, we have discerned the mechanism of starch-induced Sus complex assembly in live anaerobic cells with nanometer-scale resolution. Our insights into the starch-induced outer membrane protein assembly central to this conserved nutrient uptake mechanism pave the way for the development of dietary or pharmaceutical therapies to control Bacteroidetes in the intestinal tract to enhance human health and treat disease.

DEAD-box helicases are conserved enzymes involved in nearly all aspects of RNA metabolism, but their mechanisms of action remain unclear. Here, we investigated the mechanism of the DEAD-box protein Mss116 on its natural substrate, the group II intron ai5γ. Group II introns are structurally complex catalytic RNAs considered evolutionarily related to the eukaryotic spliceosome, and an interesting paradigm for large RNA folding. We used single-molecule fluorescence to monitor the effect of Mss116 on folding dynamics of a minimal active construct, ai5γ–D135. The data show that Mss116 stimulates dynamic sampling between states along the folding pathway, an effect previously observed only with high Mg2+ concentrations. Furthermore, the data indicate that Mss116 promotes folding through discrete ATP-independent and ATP-dependent steps. We propose that Mss116 stimulates group II intron folding through a multi-step process that involves electrostatic stabilization of early intermediates and ATP hydrolysis during the final stages of native state assembly.

The spliceosome catalyzes precursor-mRNA splicing in all eukaryotes. It consists of over 100 proteins and five small nuclear RNAs (snRNAs), including U2 and U6 snRNAs, which are essential for catalysis. Human and yeast snRNAs share structural similarities despite the fact that human snRNAs contain numerous post-transcriptional modifications. Although functions for these modifications have been proposed, their exact roles are still not well understood. To help elucidate these roles in pre-mRNA splicing, we have used single-molecule fluorescence to characterize the effect of several post-transcriptional modifications in U2 snRNA on the conformation and dynamics of the U2-U6 complex in vitro. Consistent with yeast, the human U2-U6 complex reveals the presence of a magnesium-dependent dynamic equilibrium among three conformations. Interestingly, our data show that modifications in human U2 stem I modulate the dynamic equilibrium of the U2-U6 complex by stabilizing the four-helix structure. However, the small magnitude of this effect suggests that post-transcriptional modifications in human snRNAs may have a primary role in the mediation of specific RNA-protein interactions in vivo.

By using cryo-electron microscopy, expanded 80S-like poliovirus virions (poliovirions) were visualized in complexes with four 80S-specific camelid VHHs (Nanobodies). In all four complexes, the VHHs bind to a site on the top surface of the capsid protein VP3, which is hidden in the native virus. Interestingly, although the four VHHs bind to the same site, the structures of the expanded virus differ in detail in each complex, suggesting that each of the Nanobodies has sampled a range of low-energy structures available to the expanded virion. By stabilizing unique structures of expanded virions, VHH binding permitted a more detailed view of the virus structure than was previously possible, leading to a better understanding of the expansion process that is a critical step in infection. It is now clear which polypeptide chains become disordered and which become rearranged. The higher resolution of these structures also revealed well-ordered conformations for the EF loop of VP2, the GH loop of VP3, and the N-terminal extensions of VP1 and VP2, which, in retrospect, were present in lower-resolution structures but not recognized. These structural observations help to explain preexisting mutational data and provide insights into several other stages of the poliovirus life cycle, including the mechanism of receptor-triggered virus expansion.IMPORTANCE When poliovirus infects a cell, it undergoes a change in its structure in order to pass RNA through its protein coat, but this altered state is short-lived and thus poorly understood. The structures of poliovirus bound to single-domain antibodies presented here capture the altered virus in what appear to be intermediate states. A careful analysis of these structures lets us better understand the molecular mechanism of infection and how these changes in the virus lead to productiveinfection events.KEYWORDS 80S, VHH, cryo-electron microscopy, expanded virus, poliovirus, singledomain antibodies, three-dimensional structure P oliovirus (PV) is a small, nonenveloped, positive-sense, single-stranded RNA (ssRNA) virus that is a member of the enterovirus genus of the picornavirus family (1, 2). The virus consists of an RNA genome that is surrounded by a protein shell. The shell is formed by an icosahedrally symmetric arrangement of 60 copies each of 3 large proteins (VP1, VP2, and VP3), each of which has a wedge-shaped beta barrel core (3), and the small myristoylated protein VP4 (4), which is located on the inner surface of the capsid. To infect its natural host (human epithelial cells in the gut), poliovirus must solve the problem of getting its genome through the host's protective cellular membrane. Details of how this is accomplished are now gradually emerging (5, 6).

DEAD-box helicases are conserved enzymes involved in nearly all aspects of RNA metabolism, but their mechanisms of action remain unclear. Here, we investigated the mechanism of the DEAD-box protein Mss116 on its natural substrate, the group II intron ai5γ. Group II introns are structurally complex catalytic RNAs considered evolutionarily related to the eukaryotic spliceosome, and an interesting paradigm for large RNA folding. We used single-molecule fluorescence to monitor the effect of Mss116 on folding dynamics of a minimal active construct, ai5γ-D135. The data show that Mss116 stimulates dynamic sampling between states along the folding pathway, an effect previously observed only with high Mg 2+ concentrations. Furthermore, the data indicate that Mss116 promotes folding through discrete ATP-independent and ATPdependent steps. We propose that Mss116 stimulates group II intron folding through a multi-step process that involves electrostatic stabilization of early intermediates and ATP hydrolysis during the final stages of native state assembly. KeywordsGroup II introns; Mss116 DEAD-box protein; single-molecule fluorescence DEAD-box proteins are enzymes that play essential roles in cellular processes involving RNA 1 . Although these have been studied in vitro and in vivo, there are few examples of DEAD-box proteins whose mechanisms have been dissected using a natural substrate in vitro 2 . Mss116 is a DEAD-box protein that facilitates splicing of all Saccharomyces cerevisiae (Sc.) mitochondrial group I and II introns in vivo 3 . Mss116 exhibits RNA binding, unwinding, annealing and ATPase activities and has been shown to facilitate group II intron splicing in vitro under near-physiological conditions 4-6 .Unwinding assay 250 nM Mss116 protein was pre-incubated with 0.1 nM end-labeled RNA duplex (12 bp) at 30°C for 10 min and unwinding reaction was initiated by the addition of ATP as described 4 . The unwinding reaction was performed in 100 mM KCl, 40 mM MOPS, pH 7.5, 4 mM MgCl 2 , 0.01% IGEPAL, 2 mM DTT, 2U/µL RNase inhibitor and 4 mM ATP. The sample aliquots were removed at 0 and 30 min time points and detected using 10% native PAGE. ATPase assay250 nM Mss116 protein was preincubated with or wthout 5 µM single-stranded RNA at 30°C for 10 min and all reactions were initiated by the addition of a mixture of P-32 labeled and unlabeled ATP. ATPase assays were performed according to standard protocol 35 in 100 mM KCl, 40 mM MOPS, pH 7.5, 1 mM MgCl 2 , 2 mM DTT, 2U/µL RNase inhibitor and 1 mM ATP.

Over the past decade, single-molecule fluorescence studies have elucidated the structure-function relationship of RNA molecules. The real-time observation of individual RNAs by single-molecule fluorescence has unveiled the dynamic behavior of complex RNA systems in unprecedented detail, revealing the presence of transient intermediate states and their kinetic pathways. This review provides an overview of how single-molecule fluorescence has been used to explore the dynamics of RNA folding and catalysis.