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1.  Structural basis for exon recognition by a group II intron 
Nature structural & molecular biology  2008;15(11):1221-1222.
Free group II introns are infectious retroelements that can bind and insert themselves into RNA and DNA molecules via reverse-splicing. Here we report the 3.4Å crystal structure of a complex between an oligonucleotide target substrate and a group IIC intron, as well as the refined free intron structure. The structure of the complex reveals the conformation of motifs involved in exon recognition by group II introns.
doi:10.1038/nsmb.1509
PMCID: PMC2735234  PMID: 18953333
2.  The tertiary structure of group II introns: implications for biological function and evolution 
Group II introns are some of the largest ribozymes in nature, and they are a major source of information about RNA assembly and tertiary structural organization. These introns are of biological significance because they are self-splicing mobile elements that have migrated into diverse genomes and played a major role in the genomic organization and metabolism of most life forms. The tertiary structure of group II introns has been the subject of many phylogenetic, genetic, biochemical and biophysical investigations, all of which are consistent with the recent crystal structure of an intact group IIC intron from the alkaliphilic eubacterium Oceanobacillus iheyensis. The crystal structure reveals that catalytic intron domain V is enfolded within the other intronic domains through an elaborate network of diverse tertiary interactions. Within the folded core, DV adopts an activated conformation that readily binds catalytic metal ions and positions them in a manner appropriate for reaction with nucleic acid targets. The tertiary structure of the group II intron reveals new information on motifs for RNA architectural organization, mechanisms of group II intron catalysis, and the evolutionary relationships among RNA processing systems. Guided by the structure and the wealth of previous genetic and biochemical work, it is now possible to deduce the probable location of DVI and the site of additional domains that contribute to the function of the highly derived group IIB and IIA introns.
doi:10.3109/10409231003796523
PMCID: PMC4408542  PMID: 20446804
RNA splicing; transposition; ribozyme; enzymology; crystallography
3.  Parts, assembly and operation of the RIG-I family of motors 
Host cell invasion is monitored by a series of pattern recognition receptors (PRRs) that activate the innate immune machinery upon detection of a cognate pathogen associated molecular pattern (PAMP). The RIG-I like receptor (RLR) family of PRRs includes three proteins — RIG-I, MDA5, and LGP2 — responsible for the detection of intracellular pathogenic RNA. All RLR proteins are built around an ATPase core homologous to those found in canonical Superfamily 2 (SF2) RNA helicases, which has been modified through the addition of novel accessory domains to recognize duplex RNA. This review focuses on the structural bases for pathogen-specific dsRNA binding and ATPase activation in RLRs, differential RNA recognition by RLR family members, and implications for other duplex RNA activated ATPases, such as Dicer.
doi:10.1016/j.sbi.2013.11.011
PMCID: PMC4070197  PMID: 24878341
4.  Duplex RNA activated ATPases (DRAs) 
RNA Biology  2013;10(1):111-120.
Double-stranded RNAs are an important class of functional macromolecules in living systems. They are usually found as part of highly specialized intracellular machines that control diverse cellular events, ranging from virus replication, antiviral defense, RNA interference, to regulation of gene activities and genomic integrity. Within different intracellular machines, the RNA duplex is often found in association with specific RNA-dependent ATPases, including Dicer, RIG-I and DRH-3 proteins. These duplex RNA-activated ATPases represent an emerging group of motor proteins within the large and diverse super family 2 nucleic acid-dependent ATPases (which are historically defined as SF2 helicases). The duplex RNA-activated ATPases share characteristic molecular features for duplex RNA recognition, including motifs (e.g., motifs IIa and Vc) and an insertion domain (HEL2i), and they require double-strand RNA binding for their enzymatic activities. Proteins in this family undergo large conformational changes concomitant with RNA binding, ATP binding and ATP hydrolysis in order to achieve their functions, which include the release of signaling domains and the recruitment of partner proteins. The duplex RNA-activated ATPases represent a distinct and fascinating group of nanomechanical molecular motors that are essential for duplex RNA sensing and processing in diverse cellular pathways.
doi:10.4161/rna.22706
PMCID: PMC3590228  PMID: 23228901
ATPases; antiviral immunity; Dicer; helicase; molecular motor; RIG-I like receptors; RNAi
5.  A kinetic intermediate that regulates proper folding of a group II intron RNA 
Journal of molecular biology  2007;375(2):572-580.
Summary
The D135 group II intron ribozyme follows a unique folding pathway that is direct and appears to be devoid of kinetic traps. During the earliest stages of folding, D135 collapses slowly to a compact intermediate, and all subsequent assembly events are rapid. Collapse of intron Domain 1 (D1) has been shown to limit the rate constant for D135 folding, although the specific substructure of the D1 kinetic intermediate has not yet been identified. Employing time-resolved Nucleotide Analog Interference Mapping (NAIM), we have identified a cluster of atoms within the D1 main stem that control the rate constant for D135 collapse. Functional groups within the κ–ζ element are particularly important for this earliest stage of folding, which is intriguing given that this same motif also serves later as the docking site for catalytic Domain 5 (D5). Importantly, the κ–ζ element is shown to be a divalent ion binding pocket, indicating that this region is a Mg2+-dependent switch that initiates the cascade of D135 folding events. By measuring the Mg2+ dependence of the compaction rate constant, we conclude that the actual rate-limiting step in D1 compaction involves the formation of an unstable folding intermediate that is captured by the binding of Mg2+. This carefully orchestrated folding pathway, in which formation of an active-site docking region is early and rate-limiting, ensures proper folding of the intron core and faithful splicing. It may represent an important paradigm for the folding of large, multidomain RNA molecules.
doi:10.1016/j.jmb.2007.10.052
PMCID: PMC2735237  PMID: 18022197
ribozyme; RNA folding; catalysis; splicing; RNA structure
6.  The identification of novel RNA structural motifs using COMPADRES: an automated approach to structural discovery 
Nucleic Acids Research  2004;32(22):6650-6659.
Recurring RNA structural motifs are important sites of tertiary interaction and as such, are integral to RNA macromolecular structure. Although numerous RNA motifs have been classified and characterized, the identification of new motifs is of great interest. In this study, we discovered four new conformationally recurring motifs: the π-turn, the Ω-turn, the α-loop and the C2′-endo mediated flipped adenosine motif. Not only do they have complex and interesting structures, but they participate in contacts of high biological significance. In a first for the RNA field, new motifs were discovered by a fully automated algorithm. This algorithm, COMPADRES, utilized a reduced representation of the RNA backbone and was highly successful at discerning unique structural relationships. This study also shows that recurring RNA substructures are not necessarily accompanied by consistent primary or secondary structure.
doi:10.1093/nar/gkh1002
PMCID: PMC545444  PMID: 15608296
7.  Single-molecule analysis of Mss116-mediated group II intron folding 
Nature  2010;467(7318):935-939.
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.
doi:10.1038/nature09422
PMCID: PMC4484588  PMID: 20944626
Group II introns; Mss116 DEAD-box protein; single-molecule fluorescence
8.  Native Purification and Analysis of Long RNAs 
Methods in enzymology  2015;558:3-37.
The purification and analysis of long noncoding RNAs (lncRNAs) in vitro is a challenge, particularly if one wants to preserve elements of functional structure. Here, we describe a method for purifying lncRNAs that preserves the cotranscriptionally derived structure. The protocol avoids the misfolding that can occur during denaturation–renaturation protocols, thus facilitating the folding of long RNAs to a native-like state. This method is simple and does not require addition of tags to the RNA or the use of affinity columns. LncRNAs purified using this type of native purification protocol are amenable to biochemical and biophysical analysis. Here, we describe how to study lncRNA global compaction in the presence of divalent ions at equilibrium using sedimentation velocity analytical ultracentrifugation and analytical size-exclusion chromatography as well as how to use these uniform RNA species to determine robust lncRNA secondary structure maps by chemical probing techniques like selective 2′-hydroxyl acylation analyzed by primer extension and dimethyl sulfate probing.
doi:10.1016/bs.mie.2015.01.008
PMCID: PMC4477701  PMID: 26068736
9.  The Brace for a Growing Scaffold: Mss116 Protein Promotes RNA Folding by Stabilizing an Early Assembly Intermediate 
Journal of molecular biology  2012;422(3):347-365.
The ai5γ group II intron requires a protein cofactor to facilitate native folding in the cell. Yeast protein Mss116 greatly accelerates intron folding under near-physiological conditions both in vivo and in vitro. Although the effect of Mss116 on the kinetics of ai5γ ribozyme folding and catalysis has been extensively studied, the precise structural role and interaction sites of Mss116 have been elusive. Using Nucleotide Analog Interference Mapping to study the folding of splicing precursor constructs, we have identified specific intron functional groups that participate in Mss116-facilitated folding and we have determined their role in the folding mechanism. The data indicate that Mss116 stabilizes an early, obligate folding intermediate within intron domain 1, thereby laying the foundation for productive folding to the native state. In addition, the data reveal an important role for the IBS2 exon sequence and for the terminus of domain 6, during the folding of self-splicing group IIB intron constructs.
doi:10.1016/j.jmb.2012.05.037
PMCID: PMC4419380  PMID: 22705286
ribozyme; RNA folding; DEAD-box protein; NAIM
10.  A new way to see RNA 
Quarterly reviews of biophysics  2011;44(4):433-466.
Unlike proteins, the RNA backbone has numerous degrees of freedom (eight, if one counts the sugar pucker), making RNA modeling, structure building and prediction a multidimensional problem of exceptionally high complexity. And yet RNA tertiary structures are not infinite in their structural morphology; rather, they are built from a limited set of discrete units. In order to reduce the dimensionality of the RNA backbone in a physically reasonable way, a shorthand notation was created that reduced the RNA backbone torsion angles to two (η and θ, analogous to ϕ and ψ in proteins). When these torsion angles are calculated for nucleotides in a crystallographic database and plotted against one another, one obtains a plot analogous to a Ramachandran plot (the η/θ plot), with highly populated and unpopulated regions. Nucleotides that occupy proximal positions on the plot have identical structures and are found in the same units of tertiary structure. In this review, we describe the statistical validation of the η/θ formalism and the exploration of features within the η/θ plot. We also describe the application of the η/θ formalism in RNA motif discovery, structural comparison, RNA structure building and tertiary structure prediction. More than a tool, however, the η/θ formalism has provided new insights into RNA structure itself, revealing its fundamental components and the factors underlying RNA architectural form.
doi:10.1017/S0033583511000059
PMCID: PMC4410278  PMID: 21729350
11.  Molecular Mechanics of RNA Translocases 
Methods in enzymology  2012;511:131-147.
Historically, research on RNA helicase and translocation enzymes has seemed like a footnote to the extraordinary progress in studies on DNA-remodeling enzymes. However, during the past decade, the rising wave of activity in RNA science has engendered intense interest in the behaviors of specialized motor enzymes that remodel RNA molecules. Functional, mechanistic, and structural investigations of these RNA enzymes have begun to reveal the molecular basis for their key roles in RNA metabolism and signaling. In this chapter, we highlight the structural and mechanistic similarities among monomeric RNA translo-case enzymes, while emphasizing the many divergent characteristics that have caused this enzyme family to become one of the most important in metabolism and gene expression.
doi:10.1016/B978-0-12-396546-2.00006-1
PMCID: PMC4407658  PMID: 22713318
12.  Crystal Structure of a Self-Spliced Group II Intron 
Science (New York, N.Y.)  2008;320(5872):77-82.
Group II introns are self-splicing ribozymes that catalyze their own excision from precursor transcripts and insertion into new genetic locations. Here we report the crystal structure of an intact, self-spliced group II intron from Oceanobacillus iheyensis at 3.1 angstrom resolution. An extensive network of tertiary interactions facilitates the ordered packing of intron subdomains around a ribozyme core that includes catalytic domain V. The bulge of domain V adopts an unusual helical structure that is located adjacent to a major groove triple helix (catalytic triplex). The bulge and catalytic triplex jointly coordinate two divalent metal ions in a configuration that is consistent with a two–metal ion mechanism for catalysis. Structural and functional analogies support the hypothesis that group II introns and the spliceosome share a common ancestor.
doi:10.1126/science.1153803
PMCID: PMC4406475  PMID: 18388288
13.  The RIG-I ATPase core has evolved a functional requirement for allosteric stabilization by the Pincer domain 
Nucleic Acids Research  2014;42(18):11601-11611.
Retinoic acid-inducible gene I (RIG-I) is a pattern recognition receptor expressed in metazoan cells that is responsible for eliciting the production of type I interferons and pro-inflammatory cytokines upon detection of intracellular, non-self RNA. Structural studies of RIG-I have identified a novel Pincer domain composed of two alpha helices that physically tethers the C-terminal domain to the SF2 helicase core. We find that the Pincer plays an important role in mediating the enzymatic and signaling activities of RIG-I. We identify a series of mutations that additively decouple the Pincer motif from the ATPase core and show that this decoupling results in impaired signaling. Through enzymological and biophysical analysis, we further show that the Pincer domain controls coupled enzymatic activity of the protein through allosteric control of the ATPase core. Further, we show that select regions of the HEL1 domain have evolved to potentiate interactions with the Pincer domain, resulting in an adapted ATPase cleft that is now responsive to adjacent domains that selectively bind viral RNA.
doi:10.1093/nar/gku817
PMCID: PMC4191399  PMID: 25217590
14.  Dicer-related helicase 3 forms an obligate dimer for recognizing 22G-RNA 
Nucleic Acids Research  2014;42(6):3919-3930.
Dicer is a specialized nuclease that produces RNA molecules of specific lengths for use in gene silencing pathways. Dicer relies on the correct measurement of RNA target duplexes to generate products of specific lengths. It is thought that Dicer uses its multidomain architecture to calibrate RNA product length. However, this measurement model is derived from structural information from a protozoan Dicer, and does not account for the helicase domain present in higher organisms. The Caenorhabditis elegans Dicer-related helicase 3 (DRH-3) is an ortholog of the Dicer and RIG-I family of double-strand RNA activated ATPases essential for secondary siRNA production. We find that DRH-3 specifies 22 bp RNAs by dimerization of the helicase domain, a process mediated by ATPase activity and the N-terminal domain. This mechanism for RNA length discrimination by a Dicer family protein suggests an alternative model for RNA length measurement by Dicer, with implications for recognition of siRNA and miRNA targets.
doi:10.1093/nar/gkt1383
PMCID: PMC3973318  PMID: 24435798
15.  Visualizing the ai5γ group IIB intron 
Nucleic Acids Research  2013;42(3):1947-1958.
It has become apparent that much of cellular metabolism is controlled by large well-folded noncoding RNA molecules. In addition to crystallographic approaches, computational methods are needed for visualizing the 3D structure of large RNAs. Here, we modeled the molecular structure of the ai5γ group IIB intron from yeast using the crystal structure of a bacterial group IIC homolog. This was accomplished by adapting strategies for homology and de novo modeling, and creating a new computational tool for RNA refinement. The resulting model was validated experimentally using a combination of structure-guided mutagenesis and RNA structure probing. The model provides major insights into the mechanism and regulation of splicing, such as the position of the branch-site before and after the second step of splicing, and the location of subdomains that control target specificity, underscoring the feasibility of modeling large functional RNA molecules.
doi:10.1093/nar/gkt1051
PMCID: PMC3919574  PMID: 24203709
16.  Solving nucleic acid structures by molecular replacement: examples from group II intron studies 
Strategies for phasing nucleic acid structures by molecular replacement, using both experimental and de novo designed models, are discussed.
Structured RNA molecules are key players in ensuring cellular viability. It is now emerging that, like proteins, the functions of many nucleic acids are dictated by their tertiary folds. At the same time, the number of known crystal structures of nucleic acids is also increasing rapidly. In this context, molecular replacement will become an increasingly useful technique for phasing nucleic acid crystallographic data in the near future. Here, strategies to select, create and refine molecular-replacement search models for nucleic acids are discussed. Using examples taken primarily from research on group II introns, it is shown that nucleic acids are amenable to different and potentially more flexible and sophisticated molecular-replacement searches than proteins. These observations specifically aim to encourage future crystallographic studies on the newly discovered repertoire of noncoding transcripts.
doi:10.1107/S0907444913013218
PMCID: PMC3817690  PMID: 24189228
nucleic acid sequence homology; de novo structure design; long noncoding RNA; RNA structure; homology modeling; RCrane
17.  Defining the functional determinants for RNA surveillance by RIG-I 
EMBO Reports  2013;14(9):772-779.
Defining the functional determinants for RNA surveillance by RIG-I
This study shows that HEL2i-domain mediated scanning allows RIG-I to sense the length of RNA targets, and a short RNA duplex that binds one RIG-I molecule can stimulate ATPase activity and interferon response.
Retinoic acid-inducible gene-I (RIG-I) is an intracellular RNA sensor that activates the innate immune machinery in response to infection by RNA viruses. Here, we report the crystal structure of distinct conformations of a RIG-I:dsRNA complex, which shows that HEL2i-mediated scanning allows RIG-I to sense the length of RNA targets. To understand the implications of HEL2i scanning for catalytic activity and signalling by RIG-I, we examined its ATPase activity when stimulated by duplex RNAs of varying lengths and 5′ composition. We identified a minimal RNA duplex that binds one RIG-I molecule, stimulates robust ATPase activity, and elicits a RIG-I-mediated interferon response in cells. Our results reveal that the minimal functional unit of the RIG-I:RNA complex is a monomer that binds at the terminus of a duplex RNA substrate. This behaviour is markedly different from the RIG-I paralog melanoma differentiation-associated gene 5 (MDA5), which forms cooperative filaments.
doi:10.1038/embor.2013.108
PMCID: PMC3790051  PMID: 23897087
ATP hydrolysis; innate immunity; RNA helicase; X-ray crystallography
18.  Crystal structure of a group II intron in the pre-catalytic state 
Group II introns are self-splicing catalytic RNAs that are thought to be ancestral to the spliceosome. Here we report the 3.65 Å crystal structure of the group II intron from Oceanobacillus iheyensis in the pre-catalytic state. The structure reveals the conformation of the 5′ splice site in the catalytic core and represents the first structure of an intron prior to the first step of splicing.
doi:10.1038/nsmb.2270
PMCID: PMC3670821  PMID: 22484319
19.  Now on display: a gallery of group II intron structures at different stages of catalysis 
Mobile DNA  2013;4:14.
Group II introns are mobile genetic elements that self-splice and retrotranspose into DNA and RNA. They are considered evolutionary ancestors of the spliceosome, the ribonucleoprotein complex essential for pre-mRNA processing in higher eukaryotes. Over a 20-year period, group II introns have been characterized first genetically, then biochemically, and finally by means of X-ray crystallography. To date, 17 crystal structures of a group II intron are available, representing five different stages of the splicing cycle. This review provides a framework for classifying and understanding these new structures in the context of the splicing cycle. Structural and functional implications for the spliceosome are also discussed.
doi:10.1186/1759-8753-4-14
PMCID: PMC3669008  PMID: 23634971
Retrotransposition; Spliceosome; X-ray structures; RNA catalysis; Metal ions
20.  Visualizing group II intron catalysis through the stages of splicing 
Cell  2012;151(3):497-507.
SUMMARY
Group II introns are self-splicing ribozymes that share a reaction mechanism and a common ancestor with the eukaryotic spliceosome, thereby providing a model system for understanding the chemistry of pre-mRNA splicing. Here we report fourteen crystal structures of a group II intron at different stages of catalysis. We provide a detailed mechanism for the first step of splicing, we describe a reversible conformational change between the first and the second steps of splicing, and we present the ligand-free intron structure after splicing, in an active state that corresponds to the retrotransposable form of the intron. During each reaction, the reactants are aligned and activated by a heteronuclear four-metal-ion center that contains a metal cluster and obligate monovalent cations, adopting a structural arrangement similar to that of protein endonucleases. Based on our data, we propose a model for the splicing cycle and show that it is applicable to the eukaryotic spliceosome.
doi:10.1016/j.cell.2012.09.033
PMCID: PMC3628766  PMID: 23101623
21.  Discrete RNA libraries from pseudo-torsional space 
Journal of molecular biology  2012;421(1):6-26.
The discovery that RNA molecules can fold into complex structures and carry out diverse cellular roles has led to interest in developing tools for modeling RNA tertiary structure. While significant progress has been made in establishing that the RNA backbone is rotameric, few libraries of discrete conformations specifically for use in RNA modeling have been validated. Here, we present six libraries of discrete RNA conformations based on a simplified pseudo-torsional notation of the RNA backbone, comparable to phi and psi in the protein backbone. We evaluate the ability of each library to represent single nucleotide backbone conformations and we show how individual library fragments can be assembled into dinucleotides that are consistent with established RNA backbone descriptors spanning from sugar to sugar. We then use each library to build all-atom models of 20 test folds and we show how the composition of a fragment library can limit model quality. Despite the limitations inherent in using discretized libraries, we find that several hundred discrete fragments can rebuild RNA folds up to 174 nucleotides in length with atomic-level accuracy (<1.5Å RMSD). We anticipate the libraries presented here could easily be incorporated into RNA structural modeling, analysis, or refinement tools.
doi:10.1016/j.jmb.2012.03.002
PMCID: PMC3422622  PMID: 22425640
RNA structure; RNA backbone conformation; RNA fragment library; RNA modeling
22.  The Thermodynamic Basis for Viral RNA Detection by the RIG-I Innate Immune Sensor 
The Journal of Biological Chemistry  2012;287(51):42564-42573.
Background: RIG-I is an essential innate immune receptor that detects viral RNAs in infected cells.
Results: RIG-I uses distinct subdomains to recognize specific characteristics of viral RNAs.
Conclusion: The 5′-triphosphate is critical for high affinity RIG-I/RNA interaction.
Significance: Characterizing the RIG-I/RNA interface is essential for understanding early stages of immune response against RNA viruses.
RIG-I is a cytoplasmic surveillance protein that contributes to the earliest stages of the vertebrate innate immune response. The protein specifically recognizes 5′-triphosphorylated RNA structures that are released into the cell by viruses, such as influenza and hepatitis C. To understand the energetic basis for viral RNA recognition by RIG-I, we studied the binding of RIG-I domain variants to a family of dsRNA ligands. Thermodynamic analysis revealed that the isolated RIG-I domains each make important contributions to affinity and that they interact using different strategies. Covalent linkage between the domains enhances RNA ligand specificity while reducing overall binding affinity, thereby providing a mechanism for discriminating virus from host RNA.
doi:10.1074/jbc.M112.385146
PMCID: PMC3522258  PMID: 23055530
ATPases; Interferon; RNA Helicase; RNA-Protein Interaction; Viral Immunology; RNA Triphosphate
23.  RCrane: semi-automated RNA model building 
RCrane is a new tool for the partially automated building of RNA crystallographic models into electron-density maps of low or intermediate resolution. This tool helps crystallographers to place phosphates and bases into electron density and then automatically predicts and builds the detailed all-atom structure of the traced nucleotides.
RNA crystals typically diffract to much lower resolutions than protein crystals. This low-resolution diffraction results in unclear density maps, which cause considerable difficulties during the model-building process. These difficulties are exacerbated by the lack of computational tools for RNA modeling. Here, RCrane, a tool for the partially automated building of RNA into electron-density maps of low or intermediate resolution, is presented. This tool works within Coot, a common program for macromolecular model building. RCrane helps crystallographers to place phosphates and bases into electron density and then automatically predicts and builds the detailed all-atom structure of the traced nucleotides. RCrane then allows the crystallographer to review the newly built structure and select alternative backbone conformations where desired. This tool can also be used to automatically correct the backbone structure of previously built nucleotides. These automated corrections can fix incorrect sugar puckers, steric clashes and other structural problems.
doi:10.1107/S0907444912018549
PMCID: PMC3413212  PMID: 22868764
RCrane; RNA model building
24.  Structural insights into RNA recognition by RIG-I 
Cell  2011;147(2):409-422.
Summary
Intracellular RIG-I-like receptors (RLRs, including RIG-I, MDA-5, and LGP-2) recognize viral RNAs as pathogen-associated molecular patterns (PAMPs) and initiate an antiviral immune response. To understand the molecular basis of this process, we determined the crystal structure of RIG-I in complex with double-stranded RNA. The dsRNA is sheathed within a network of protein domains that include a conserved “helicase” domain (regions HEL1 and HEL2), a specialized insertion domain (HEL2i), and a C-terminal regulatory domain (CTD). A V-shaped pincer connects HEL2 and the CTD by gripping an α-helical shaft that extends from HEL1. In this way, the pincer coordinates functions of all the domains and couples RNA binding with ATP hydrolysis. RIG-I falls within the Dicer-RIG-I clade of super family 2 of helicases and this structure reveals complex interplay between motor domains, accessory mechanical domains and RNA that has implications for understanding the nanomechanical function this protein family and other ATPases more broadly.
doi:10.1016/j.cell.2011.09.023
PMCID: PMC3222294  PMID: 22000018
RIG-I; RNA helicase; innate immunity; X-ray crystallography
25.  Mechanism of Mss116 ATPase reveals functional diversity of DEAD-box proteins 
Journal of molecular biology  2011;409(3):399-414.
Mss116 is a Saccharomyces cerevisiae mitochondrial DEAD-box RNA helicase protein essential for efficient in vivo splicing of all group I and II introns and activation of mRNA translation. Catalysis of intron splicing by Mss116 is coupled to its ATPase activity. Knowledge of the kinetic pathway(s) and biochemical intermediates populated during RNA-stimulated Mss116 ATPase is fundamental for defining how Mss116 ATP utilization is linked to in vivo function. We therefore measured the rate and equilibrium constants underlying Mss116 ATP utilization and nucleotide-linked RNA binding. RNA accelerates the Mss116 steady-state ATPase ~7-fold by promoting rate-limiting ATP hydrolysis, such that Pi release becomes (partially) rate-limiting. RNA binding displays strong thermodynamic coupling to the chemical states of the Mss116-bound nucleotide such that Mss116 with bound ADP-Pi binds RNA more strongly than with bound ADP or in the absence of nucleotide. The predominant biochemical intermediate populated during in vivo steady-state cycling is the strong RNA binding, Mss116-ADP-Pi state. Strong RNA binding allows Mss116 to fulfill its biological role in stabilization of group II intron folding intermediates. ATPase cycling allows for transient population of the weak RNA binding, ADP state of Mss116 and linked dissociation from RNA, which is required for the final stages of intron folding. In cases where Mss116 functions as a helicase, the data collectively favor a model in which ATP hydrolysis promotes a weak-to-strong RNA binding transition that disrupts stable RNA duplexes. The subsequent strong-to-weak RNA binding transition associated with Pi release dissociates RNA-Mss116 complexes, regenerating free Mss116.
doi:10.1016/j.jmb.2011.04.004
PMCID: PMC3125984  PMID: 21501623
RNA helicase; ATPase cycle; kinetics; fluorescence correlation spectroscopy (FCS)

Results 1-25 (41)