Use of duplex rigidity for stability and specificity in RNA tertiary structure

The Tetrahymena group I ribozyme's oligonucleotide substrate, CCCUCUA(5), forms six base pairs with the ribozyme's internal guide sequence (IGS, 5'GGAGGG) to give the P1 duplex, and this duplex then docks into the active site via tertiary interactions. Shortening the substrate by three residues to give UCUA(5) reduces the equilibrium constant for P1 docking by approximately 200-fold even though UCUA(5) retains all the functional groups known to be involved in tertiary interactions [Narlikar, G. J., Bartley, L. E., Khosla, M., and Herschlag, D. (1999) Biochemistry 38, 14192-14204]. Here we show that the P1 duplex formed with UCUA(5) engages in all of the major tertiary interactions made by the standard P1 duplex. This suggests that the destabilization is not due to disruption of specific tertiary interactions. It therefore appears that the weaker docking of UCUA(5) arises from the increased conformational freedom of the undocked P1 duplex, which has three unpaired IGS residues and thus a larger entropic cost for docking. Further, a 2'-methoxy substitution at an IGS residue that is base-paired in the standard P1 duplex with CCCUCUA(5) but unpaired in the P1 duplex with UCUA(5) destabilizes docking of the standard P1 duplex approximately 300-fold more than it destabilizes docking of the P1 duplex formed with UCUA(5). These results suggest that fixation of groups in the context of a rigid duplex may be a general strategy used by RNA to substantially increase interaction specificity, both by aiding binding of the desired functional groups and by increasing the energetic cost of forming alternative interactions.     

Modular construction of a tertiary RNA structure: the specificity domain of the Bacillus subtilis RNase P RNA

The structure of the specificity domain (S-domain) of the Bacillus subtilis RNase P RNA has been proposed to be composed of a core and a buttress module, analogous to the bipartite structure of the P4-P6 domain of the Tetrahymena group I ribozyme. The core module is the functional unit of the S-domain and contains the binding site for the T stem-loop of a tRNA. The buttress module provides structural stability to the core module and consists of a GA3 tetraloop and its receptor. To explicitly test the hypothesis that modular construction can describe the structure of the S-domain and is a useful RNA design strategy, we analyzed the equilibrium folding and substrate binding of three classes of S-domain mutants. Addition or deletion of a base pair in the helical linker region between the modules only modestly destabilizes the tertiary structure. tRNA binding selectivity is affected in one but not in two other mutants of this class. Elimination of the GA3 tetraloop-receptor interactions significantly destabilizes the core module and results in the loss of tRNA binding selectivity. Replacing the buttress module with that of a homologous RNase P RNA maintains the tRNA binding selectivity. Overall, we have observed that the linker regions between the two modules can tolerate moderate structural changes and that the buttress modules can be shuffled between homologous S-domains. These results suggest that it is feasible to design an RNA using a buttress module to stabilize a functional module.     

Manganese(II) as a paramagnetic probe of the tertiary structure of transfer RNA.

The effect of manganese on both the low field (10--15 ppm) and the high field (o--3 ppm) NMR spectra of unfractionated tRNA and yeast tRNAPhe has been investigated. Trace amounts of Mn2+ cause selective broadening of resonances which are assigned to specific tertiary interactions. The order in which resonances broaden is the same as the order in which they are stabilized by the addition of magnesium, namely s4U8 - A14, U33 and A58 - T54. From this we conclude that three of the strong binding sites probably are the same for both Mn2+ and Mg2+, and that these sites are located close to the tertiary interactions which are stabilized by the strongly bound metals. The broadening data, taken in conjunction with published X-ray data on yeast tRNAPhe, permit us to suggest some plausible locations for the strong binding sites.     

Energetics and cooperativity of tertiary hydrogen bonds in RNA structure.

Tertiary interactions that allow RNA to fold into intricate three-dimensional structures are being identified, but little is known about the thermodynamics of individual interactions. Here we quantify the tertiary structure contributions of individual hydrogen bonds in a "ribose zipper" motif of the recently crystallized Tetrahymena group I intron P4-P6 domain. The 2'-hydroxyls of P4-P6 nucleotides C109/A184 and A183/G110 participate in forming the "teeth" of the zipper. These four nucleotides were substituted in all combinations with their 2'-deoxy and (separately) 2'-methoxy analogues, and thermodynamic effects on the tertiary folding DeltaG degrees ' were assayed by the Mg2+ dependence of electrophoretic mobility in nondenaturing gels. The 2'-deoxy series showed a consistent trend with an average contribution to the tertiary folding DeltaG degrees' of -0.4 to -0.5 kcal/mol per hydrogen bond. Contributions were approximately additive, reflecting no cooperativity among the hydrogen bonds. Each "tooth" of the ribose zipper (comprising two hydrogen bonds) thus contributes about -1.0 kcal/mol to the tertiary folding DeltaG degrees'. Single 2'-methoxy substitutions destabilized folding by approximately 1 kcal/mol, but the trend reversed with multiple 2'-methoxy substitutions; the folding DeltaG degrees' for the quadruple 2'-methoxy derivative was approximately unchanged relative to wild-type. On the basis of these data and on temperature-gradient gel results, we conclude that entropically favorable hydrophobic interactions balance enthalpically unfavorable hydrogen bond deletions and steric clashes for multiple 2'-methoxy substitutions. Because many of the 2'-deoxy derivatives no longer have the characteristic hydrogen-bond patterns of the ribose zipper motif but simply have individual long-range ribose-base or ribose-ribose hydrogen bonds, we speculate that the energetic value of -0.4 to -0.5 kcal/mol per tertiary hydrogen bond may be more generally applicable to RNA folding.     

A range of complex probabilistic models for RNA secondary structure prediction that includes the nearest-neighbor model and more

The standard approach for single-sequence RNA secondary structure prediction uses a nearest-neighbor thermodynamic model with several thousand experimentally determined energy parameters. An attractive alternative is to use statistical approaches with parameters estimated from growing databases of structural RNAs. Good results have been reported for discriminative statistical methods using complex nearest-neighbor models, including CONTRAfold, Simfold, and ContextFold. Little work has been reported on generative probabilistic models (stochastic context-free grammars [SCFGs]) of comparable complexity, although probabilistic models are generally easier to train and to use. To explore a range of probabilistic models of increasing complexity, and to directly compare probabilistic, thermodynamic, and discriminative approaches, we created TORNADO, a computational tool that can parse a wide spectrum of RNA grammar architectures (including the standard nearest-neighbor model and more) using a generalized super-grammar that can be parameterized with probabilities, energies, or arbitrary scores. By using TORNADO, we find that probabilistic nearest-neighbor models perform comparably to (but not significantly better than) discriminative methods. We find that complex statistical models are prone to overfitting RNA structure and that evaluations should use structurally nonhomologous training and test data sets. Overfitting has affected at least one published method (ContextFold). The most important barrier to improving statistical approaches for RNA secondary structure prediction is the lack of diversity of well-curated single-sequence RNA secondary structures in current RNA databases.     

Transfer RNA splicing in Saccharomyces cerevisiae. Secondary and tertiary structures of the substrates

Secondary and tertiary structures of four yeast tRNA precursors that contain introns have been elucidated using limited digestion with a variety of single-strand- and double-strand-specific nucleases. The pre-tRNAs, representing the variety of intron sizes and potential structures, were: pre-tRNALeuCAA, pre-tRNALeuUAG, pre-tRNAIleUAU, and pre-tRNAPro-4UGG. Conventional tRNA cloverleaf structure is maintained in these precursors except that the anticodon loop is interrupted by the intron. The intron contains a sequence which is complementary to a portion of the anticodon loop and allows the formation of a double helix often extending the anticodon stem. The 5' and 3' splicing cleavage sites are located at either end of this helix and are single-stranded. The intron is the most sensitive region to nuclease cleavage, suggesting that it is on the surface of the molecule and available for interaction with the splicing endonuclease. Absence of Mg2+ or spermidine renders the dihydrouridine and T psi C loops of these precursors highly sensitive to nuclease digestion. These ionic effects mimic those observed for tRNAPhe and suggest that the tRNA portion of these precursors has native tRNA structure. We propose consensus secondary and tertiary structures which may be of significance to eventual understanding of the mechanism of yeast tRNA splicing.     

A compact RNA tertiary structure contains a buried backbone-K+ complex

The structure of a 58 nucleotide ribosomal RNA fragment buries several phosphate groups of a hairpin loop within a large tertiary core. During refinement of an X-ray crystal structure containing this RNA, a potassium ion was found to be contacted by six oxygen atoms from the buried phosphate groups; the ion is contained completely within the solvent-accessible surface of the RNA. The electrostatic potential at the ion chelation site is unusually large, and more than compensates for the substantial energetic penalties associated with partial dehydration of the ion and displacement of delocalized ions. The very large predicted binding free energy, approximately -30 kcal/mol, implies that the site must be occupied for the RNA to fold. These findings agree with previous studies of the ion-dependent folding of tertiary structure in this RNA, which concluded that a monovalent ion was bound in a partially dehydrated environment where Mg2+ could not easily compete for binding. By compensating the unfavorable free energy of buried phosphate groups with a chelated ion, the RNA is able to create a larger and more complex tertiary fold than would be possible otherwise.     

The role of RNA structure in the interaction of U1A protein with U1 hairpin II RNA

The N-terminal RNA Recognition Motif (RRM1) of the spliceosomal protein U1A interacting with its target U1 hairpin II (U1hpII) has been used as a paradigm for RRM-containing proteins interacting with their RNA targets. U1A binds to U1hpII via direct interactions with a 7-nucleotide (nt) consensus binding sequence at the 5' end of a 10-nt loop, and via hydrogen bonds with the closing C-G base pair at the top of the RNA stem. Using surface plasmon resonance (Biacore), we have examined the role of structural features of U1hpII in binding to U1A RRM1. Mutational analysis of the closing base pair suggests it plays a minor role in binding and mainly prevents "breathing" of the loop. Lengthening the stem and nontarget part of the loop suggests that the increased negative charge of the RNA might slightly aid association. However, this is offset by an increase in dissociation, which may be caused by attraction of the RRM to nontarget parts of the RNA. Studies of a single stranded target and RNAs with untethered loops indicate that structure is not very relevant for association but is important for complex stability. In particular, breaking the link between the stem and the 5' side of the loop greatly increases complex dissociation, presumably by hindering simultaneous contacts between the RRM and stem and loop nucleotides. While binding of U1A to a single stranded target is much weaker than to U1hpII, it occurs with nanomolar affinity, supporting recent evidence that binding of unstructured RNA by U1A has physiological significance.     

Influenza A virus coding regions exhibit host-specific global ordered RNA structure

Influenza A is a significant public health threat, partially because of its capacity to readily exchange gene segments between different host species to form novel pandemic strains. An understanding of the fundamental factors providing species barriers between different influenza hosts would facilitate identification of strains capable of leading to pandemic outbreaks and could also inform vaccine development. Here, we describe the difference in predicted RNA secondary structure stability that exists between avian, swine and human coding regions. The results predict that global ordered RNA structure exists in influenza A segments 1, 5, 7 and 8, and that ranges of free energies for secondary structure formation differ between host strains. The predicted free energy distributions for strains from avian, swine, and human species suggest criteria for segment reassortment and strains that might be ideal candidates for viral attenuation and vaccine development.     

A universal small molecule,inorganic phosphate,restricts the substrate specificity of Dicer-2 in small RNA biogenesis

The enzyme Dicer is central to the production of small silencing RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). Like other insects, Drosophila melanogaster uses different Dicers to make siRNAs and miRNAs: Dicer-1 produces miRNAs from pre-miRNAs, whereas Dicer-2 generates siRNAs from long double-stranded RNA (dsRNA). How do the 2 Dicers achieve their substrate specificity? Here, we review recent findings that inorganic phosphate restricts the substrate specificity of Dicer-2 to long dsRNA. Inorganic phosphate inhibits Dicer-2 from binding and cleaving pre-miRNAs, without affecting the processing of long dsRNA. Crystal structures of a fragment of human Dicer in complex with an RNA duplex identify a phosphate-binding pocket that recognizes both the 5′-monophosphate of a substrate RNA and inorganic phosphate. We propose that inorganic phosphate occupies the phosphate-binding pocket in the fly Dicer-2, blocking binding of pre-miRNA and restricting pre-miRNA processing to Dicer-1. Thus, a small molecule can alter the substrate specificity of a nucleic acid-processing enzyme.     

Analyses of SELEX-derived ZAP-binding RNA aptamers suggest that the binding specificity is determined by both structure and sequence of the RNA

The zinc-finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses, including murine leukemia virus, Sindbis virus and Ebola virus, by targeting the viral mRNAs for degradation. ZAP directly binds to the target viral mRNA and recruits the cellular RNA degradation machinery to degrade the RNA. No significant sequence similarity or obvious common motifs have been found in the so far identified target viral mRNAs. The minimum length of the target sequence is about 500nt long. Short workable ZAP-binding RNAs should facilitate further studies on the ZAP-RNA interaction and characterization of such RNAs may provide some insights into the underlying mechanism. In this study, we used the SELEX method to isolate ZAP-binding RNA aptamers. After 21 rounds of selection, ZAP-binding aptamers were isolated. Sequence analysis revealed that they are G-rich RNAs with predicted stem-loop structures containing conserved “GGGUGG” and “GAGGG” motifs in the loop region. Insertion of the aptamer sequence into a luciferase reporter failed to render the reporter sensitive to ZAP. However, overexpression of the aptamers modestly but significantly reduced ZAP’s antiviral activity. Substitution of the conserved motifs of the aptamers significantly impaired their ZAP-binding ability and ZAP-antagonizing activity, suggesting that the RNA sequence is important for specific interaction between ZAP and the target RNA. The aptamers identified in this report should provide useful tools to further investigate the details of the interaction between ZAP and the target RNAs.     

Probing RNA tertiary structure: interhelical crosslinking of the hammerhead ribozyme.

Distinct structural models for the hammerhead ribozyme derived from single-crystal X-ray diffraction and fluorescence resonance energy transfer (FRET) measurements have been compared. Both models predict the same overall geometry, a wishbone shape with helices II and III nearly colinear and helix I positioned close to helix II. However, the relative orientations of helices I and II are different. To establish whether one of the models represents a kinetically active structure, a new crosslinking procedure was developed in which helices I and II of hammerhead ribozymes were disulfide-crosslinked via the 2' positions of specific sugar residues. Crosslinking residues on helices I and II that are close according to the X-ray structure did not appreciably reduce the catalytic efficiency. In contrast, crosslinking residues closely situated according to the FRET model dramatically reduced the cleavage rate by at least three orders of magnitude. These correlations between catalytic efficiencies and spatial proximities are consistent with the X-ray structure.     

Automated extraction and classification of RNA tertiary structure cyclic motifs

A minimum cycle basis of the tertiary structure of a large ribosomal subunit (LSU) X-ray crystal structure was analyzed. Most cycles are small, as they are composed of 3- to 5 nt, and repeated across the LSU tertiary structure. We used hierarchical clustering to quantify and classify the 4 nt cycles. One class is defined by the GNRA tetraloop motif. The inspection of the GNRA class revealed peculiar instances in sequence. First is the presence of UA, CA, UC and CC base pairs that substitute the usual sheared GA base pair. Second is the revelation of GNR(X(n))A tetraloops, where X(n) is bulged out of the classical GNRA structure, and of GN/RA formed by the two strands of interior-loops. We were able to unambiguously characterize the cycle classes using base stacking and base pairing annotations. The cycles identified correspond to small and cyclic motifs that compose most of the LSU RNA tertiary structure and contribute to its thermodynamic stability. Consequently, the RNA minimum cycles could well be used as the basic elements of RNA tertiary structure prediction methods.     

Identification of potential conserved RNA secondary structure throughout influenza A coding regions

Influenza A is a negative sense RNA virus of significant public health concern. While much is understood about the life cycle of the virus, knowledge of RNA secondary structure in influenza A virus is sparse. Predictions of RNA secondary structure can focus experimental efforts. The present study analyzes coding regions of the eight viral genome segments in both the (+) and (-) sense RNA for conserved secondary structure. The predictions are based on identifying regions of unusual thermodynamic stabilities and are correlated with studies of suppression of synonymous codon usage (SSCU). The results indicate that secondary structure is favored in the (+) sense influenza RNA. Twenty regions with putative conserved RNA structure have been identified, including two previously described structured regions. Of these predictions, eight have high thermodynamic stability and SSCU, with five of these corresponding to current annotations (e.g., splice sites), while the remaining 12 are predicted by the thermodynamics alone. Secondary structures with high conservation of base-pairing are proposed within the five regions having known function. A combination of thermodynamics, amino acid and nucleotide sequence comparisons along with SSCU was essential for revealing potential secondary structures.     

A multiprotein complex that interacts with RNA polymerase II elongator

A three-subunit Hap complex that interacts with the RNA polymerase II Elongator was isolated from yeast. Deletions of genes for two Hap subunits, HAP1 and HAP3, confer pGKL killer-insensitive and weak Elongator phenotypes. Preferential interaction of the Hap complex with free rather than RNA polymerase II-associated Elongator suggests a role in the regulation of Elongator activity.     

A novel tertiary interaction in M1 RNA, the catalytic subunit of Escherichia coli RNase P.

Phylogenetic covariation of the nucleotides corresponding to the bases at positions 121 and 236 in Escherichia coli RNase P RNA (M1 RNA) has been demonstrated in eubacterial RNase P RNAs. To investigate whether the nucleotides at these positions interact in M1 RNA we introduced base substitutions at either or at both of these positions. Single base substitutions at 121 or at 236 resulted in M1 RNA molecules which did not complement the temperature-sensitive phenotype associated with rnpA49 in vivo whereas wild-type M1 RNA or the double mutant M1 RNA, with restored base-pairing between 121 and 236, did. In addition, wild-type and the double mutant M1 RNA were efficiently cleaved by Pb++ between positions 122 and 123 whereas the rate of this cleavage was significantly reduced for the singly mutated M1 RNA variants. From these data we conclude that the nucleotides at positions 121 and 236 in M1 RNA establish a novel long-range tertiary interaction in M1 RNA. Our results also demonstrated that this interaction is not absolutely required for cleavage in vitro, however, a disruption resulted in a reduction in cleavage efficiency (kcat/Km), both in the absence and presence of C5.     

In vitro RNA synthesis from exogenous dengue viral RNA templates requires long range interactions between 5'- and 3'-terminal regions that influence RNA structure

Viral replicases of many positive-strand RNA viruses are membrane-bound complexes of cellular and viral proteins that include viral RNA-dependent RNA polymerase (RdRP). The in vitro RdRP assay system that utilizes cytoplasmic extracts from dengue viral-infected cells and exogenous RNA templates was developed to understand the mechanism of viral replication in vivo. Our results indicated that in vitro RNA synthesis at the 3'-untranslated region (UTR) required the presence of the 5'-terminal region (TR) and the two cyclization (CYC) motifs suggesting a functional interaction between the TRs. In this study, using a psoralen-UV cross-linking method and an in vitro RdRP assay, we analyzed structural determinants for physical and functional interactions. Exogenous RNA templates that were used in the assays contained deletion mutations in the 5'-TR and substitution mutations in the 3'-stem-loop structure including those that would disrupt the predicted pseudoknot structure. Our results indicate that there is physical interaction between the 5'-TR and 3'-UTR that requires only the CYC motifs. RNA synthesis at the 3'-UTR, however, requires long range interactions involving the 5'-UTR, CYC motifs, and the 3'-stem-loop region that includes the tertiary pseudoknot structure.