Structure-function relationships in the hammerhead ribozyme probed by base rescue.

We previously showed that the deleterious effects from introducing abasic nucleotides in the hammerhead ribozyme core can, in some instances, be relieved by exogenous addition of the ablated base and that the relative ability of different bases to rescue catalysis can be used to probe functional aspects of the ribozyme structure [Peracchi et al., Proc NatAcad Sci USA 93:11522]. Here we examine rescue at four additional positions, 3, 9, 12 and 13, to probe transition state interactions and to demonstrate the strengths and weaknesses of base rescue as a tool for structure-function studies. The results confirm functional roles for groups previously probed by mutagenesis, provide evidence that specific interactions observed in the ground-state X-ray structure are maintained in the transition state, and suggest formation in the transition state of other interactions that are absent in the ground state. In addition, the results suggest transition state roles for some groups that did not emerge as important in previous mutagenesis studies, presumably because base rescue has the ability to reveal interactions that are obscured by local structural redundancy in traditional mutagenesis. The base rescue results are complemented by comparing the effects of the abasic and phenyl nucleotide substitutions. The results together suggest that stacking of the bases at positions 9, 13 and 14 observed in the ground state is important for orienting other groups in the transition state. These findings add to our understanding of structure-function relationships in the hammerhead ribozyme and help delineate positions that may undergo rearrangements in the active hammerhead structure relative to the ground-state structure. Finally, the particularly efficient rescue by 2-methyladenine at position 13 relative to adenine and other bases suggests that natural base modifications may, in some instance, provide additional stability by taking advantage of hydrophobic interactions in folded RNAs.     

Towards a DNA-like duplex without hydrogen bonds

The inverse quadrupolar moments of the phenyl and pentafluorophenyl residues in the base pair P-F5 promotes strong intramolecular stacking interactions in DNA duplexes. The more natural base pairs are replaced by this novel pair the higher the thermodynamic stability of the resulting duplex if they are arranged in an alternating fashion.     

Stacking interactions in PUF-RNA complexes

Stacking interactions between amino acids and bases are common in RNA-protein interactions. Many proteins that regulate mRNAs interact with single-stranded RNA elements in the 3' UTR (3'-untranslated region) of their targets. PUF proteins are exemplary. Here we focus on complexes formed between a Caenorhabditis elegans PUF protein, FBF, and its cognate RNAs. Stacking interactions are particularly prominent and involve every RNA base in the recognition element. To assess the contribution of stacking interactions to formation of the RNA-protein complex, we combine in vivo selection experiments with site-directed mutagenesis, biochemistry, and structural analysis. Our results reveal that the identities of stacking amino acids in FBF affect both the affinity and specificity of the RNA-protein interaction. Substitutions in amino acid side chains can restrict or broaden RNA specificity. We conclude that the identities of stacking residues are important in achieving the natural specificities of PUF proteins. Similarly, in PUF proteins engineered to bind new RNA sequences, the identity of stacking residues may contribute to "target" versus "off-target" interactions, and thus be an important consideration in the design of proteins with new specificities.     

Towards a DNA-Like Duplex Without Hydrogen Bonds

Abstract

The inverse quadrupolar moments of the phenyl and pentafluorophenyl residues in the base pair P-F⁵ promotes strong intramolecular stacking interactions in DNA duplexes. The more natural base pairs are replaced by this novel pair the higher the thermodynamic stability of the resulting duplex if they are arranged in an alternating fashion.     

ClaRNA: a classifier of contacts in RNA 3D structures based on a comparative analysis of various classification schemes

The understanding of folding and function of RNA molecules depends on the identification and classification of interactions between ribonucleotide residues. We developed a new method named ClaRNA for computational classification of contacts in RNA 3D structures. Unique features of the program are the ability to identify imperfect contacts and to process coarse-grained models. Each doublet of spatially close ribonucleotide residues in a query structure is compared to clusters of reference doublets obtained by analysis of a large number of experimentally determined RNA structures, and assigned a score that describes its similarity to one or more known types of contacts, including pairing, stacking, base–phosphate and base–ribose interactions. The accuracy of ClaRNA is 0.997 for canonical base pairs, 0.983 for non-canonical pairs and 0.961 for stacking interactions. The generalized squared correlation coefficient (GC²) for ClaRNA is 0.969 for canonical base pairs, 0.638 for non-canonical pairs and 0.824 for stacking interactions. The classifier can be easily extended to include new types of spatial relationships between pairs or larger assemblies of nucleotide residues. ClaRNA is freely available via a web server that includes an extensive set of tools for processing and visualizing structural information about RNA molecules.     

An aspartate transcarbamylase lacking catalytic subunit interactions. Study of conformational changes by ultraviolet absorbance and circular dichroism spectroscopy.

A modified form of aspartate transcarbamylase is synthesized by Escherichia coli in the presence of 2-thiouracil which does not exhibit homotropic cooperative interactions between active sites yet retains heterotropic cooperative interactions due to nucleotide binding. The conformational changes induced in the modified enzyme by the binding of different ligands (substrates, substrate analogs, a transition state analog, and nucleotide effectors) were studied using ultraviolet absorbance and circular dichroism difference spectroscopy. Comparison of the results for the modified enzyme and its isolated subunits to those for the native enzyme and its isolated subunits showed that the conformational changes detected by these methods are qualitatively similar in the two enzymes. Comparison of the absorbance difference spectra due to the binding of a transition substrate analog to the intact native or modified enzymes to the corresponding results for the isolated subunits suggested that ligand binding causes an increased exposure to solvent of certain tyrosyl and phenylalanyl residues in the intact enzymes but not in the isolated subunits. This result is consistent with a diminution of subunit contacts due to substrate binding in the course of homotropic interactions in the native enzyme. Such conformational changes, though perhaps necessary for homotropic cooperativity, are not sufficient to cause homotropic cooperativity since the modified enzyme gave identical perturbations. Interactions of the transition state analog, N-(phosphonacetyl)-L-aspartate, with the modified enzyme were studied. Enzyme kinetic data obtained at low aspartate concentrations showed that this transition state analog does not stimulate activity, but rather exhibits the inhibition predicted for the total absence of homotropic cooperative interactions in the modified enzyme. Spectrophotometric titrations of the number of catalytic sites with the transition state analog showed that the modified enzyme and its isolated subunits possess, respectively, four and two high affinity sites for the inhibitor instead of six and three observed in the case of the normal enzyme and its isolated catalytic subunits. These results are correlated with the lower specific enzymatic activities of the modified enzyme and its catalytic subunits compared to the normal corresponding enzymatic species.     

Solution structure of a GAAG tetraloop in helix 6 of SRP RNA from Pyrococcus furiosus

The NMR structure of a 12-mer RNA derived from the helix 6 of SRP RNA from Pyrococcus furiosus, whose loop-closing base pair is U.G, was determined, and the structural and thermodynamic properties of the RNA were compared with those of a mutant RNA with the C:G closing base pair. Although the structures of the two RNAs are similar to each other and adopt the GNRR motif the conformational stabilities are significantly different to each other It was suggested that weaker stacking interaction of the GAAG loop with the U:G closing base pair in 12-mer RNA causes the lower conformational stability.     

Using NMR and molecular dynamics to link structure and dynamics effects of the universal base 8-aza, 7-deaza,N8 linked adenosine analog

A truly universal nucleobase enables a host of novel applications such as simplified templates for PCR primers, randomized sequencing and DNA based devices. A universal base must pair indiscriminately to each of the canonical bases with little or preferably no destabilization of the overall duplex. In reality, many candidates either destabilize the duplex or do not base pair indiscriminatingly. The novel base 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin- 4-amine) N⁸-(2′deoxyribonucleoside), a deoxyadenosine analog (UB), pairs with each of the natural DNA bases with little sequence preference. We have utilized NMR complemented with molecular dynamic calculations to characterize the structure and dynamics of a UB incorporated into a DNA duplex. The UB participates in base stacking with little to no perturbation of the local structure yet forms an unusual base pair that samples multiple conformations. These local dynamics result in the complete disappearance of a single UB proton resonance under native conditions. Accommodation of the UB is additionally stabilized via heightened backbone conformational sampling. NMR combined with various computational techniques has allowed for a comprehensive characterization of both structural and dynamic effects of the UB in a DNA duplex and underlines that the UB as a strong candidate for universal base applications.     

RNA modifications stabilize the tertiary structure of tRNAfMet by locally increasing conformational dynamics

A plethora of modified nucleotides extends the chemical and conformational space for natural occurring RNAs. tRNAs constitute the class of RNAs with the highest modification rate. The extensive modification modulates their overall stability, the fidelity and efficiency of translation. However, the impact of nucleotide modifications on the local structural dynamics is not well characterized. Here we show that the incorporation of the modified nucleotides in tRNA^fMet from Escherichia coli leads to an increase in the local conformational dynamics, ultimately resulting in the stabilization of the overall tertiary structure. Through analysis of the local dynamics by NMR spectroscopic methods we find that, although the overall thermal stability of the tRNA is higher for the modified molecule, the conformational fluctuations on the local level are increased in comparison to an unmodified tRNA. In consequence, the melting of individual base pairs in the unmodified tRNA is determined by high entropic penalties compared to the modified. Further, we find that the modifications lead to a stabilization of long-range interactions harmonizing the stability of the tRNA’s secondary and tertiary structure. Our results demonstrate that the increase in chemical space through introduction of modifications enables the population of otherwise inaccessible conformational substates.     

Solution Structure of a GAAG Tetraloop in Helix 6 of SRP RNA from Pyrococcus furiosus

The NMR structure of a 12-mer RNA derived from the helix 6 of SRP RNA from Pyrococcus furiosus, whose loop-closing base pair is U:G, was determined, and the structural and thermodynamic properties of the RNA were compared with those of a mutant RNA with the C:G closing base pair. Although the structures of the two RNAs are similar to each other and adopt the GNRR motif, the conformational stabilities are significantly different to each other. It was suggested that weaker stacking interaction of the GAAG loop with the U:G closing base pair in 12-mer RNA causes the lower conformational stability.     

Prevalence of syn nucleobases in the active sites of functional RNAs

Biological RNAs, like their DNA counterparts, contain helical stretches, which have standard Watson-Crick base pairs in the anti conformation. Most functional RNAs also adopt geometries with far greater complexity such as bulges, loops, and multihelical junctions. Occasionally, nucleobases in these regions populate the syn conformation wherein the base resides close to or over the ribose sugar, which leads to a more compact state. The importance of the syn conformation to RNA function is largely unknown. In this study, we analyze 51 RNAs with tertiary structure, including aptamers, riboswitches, ribozymes, and ribosomal RNAs, for number, location, and properties of syn nucleobases. These RNAs represent the set of nonoverlapping, moderate- to high-resolution structures available at present. We find that syn nucleobases are much more common among purines than pyrimidines, and that they favor C2'-endo-like conformations especially among those nucleobases in the intermediate syn conformation. Strikingly, most syn nucleobases participate in tertiary stacking and base-pairing interactions: Inspection of RNA structures revealed that the majority of the syn nucleobases are in regions assigned to function, with many syn nucleobases interacting directly with a ligand or ribozyme active site. These observations suggest that judicious placement of conformationally restricted nucleotides biased into the syn conformation could enhance RNA folding and catalysis. Such changes could also be useful for locking RNAs into functionally competent folds for use in X-ray crystallography and NMR.     

Solution structure of a GAAA tetraloop receptor RNA.

The GAAA tetraloop receptor is an 11-nucleotide RNA sequence that participates in the tertiary folding of a variety of large catalytic RNAs by providing a specific binding site for GAAA tetraloops. Here we report the solution structure of the isolated tetraloop receptor as solved by multidimensional, heteronuclear magnetic resonance spectroscopy. The internal loop of the tetraloop receptor has three adenosines stacked in a cross-strand or zipper-like fashion. This arrangement produces a high degree of base stacking within the asymmetric internal loop without extrahelical bases or kinking the helix. Additional interactions within the internal loop include a U. U mismatch pair and a G.U wobble pair. A comparison with the crystal structure of the receptor RNA bound to its tetraloop shows that a conformational change has to occur upon tetraloop binding, which is in good agreement with previous biochemical data. A model for an alternative binding site within the receptor is proposed based on the NMR structure, phylogenetic data and previous crystallographic structures of tetraloop interactions.     

Changes in the conformation and stability of 5 S RNA upon the binding of ribosomal proteins.

The binding of ribosomal proteins L25, L18, and L5 to 5 S RNA results in a conformational change and a destabilization of the 5 S RNA molecule. The changes observed in the near ultraviolet circular dichroism (CD) spectra and in the melting profiles indicate an increase in base stacking uith an accompanying increase in asymmetry of the bases and a decrease in the conformational stability of the 5 S RNA. These results are consistent with the interpretation that the binding of these proteins increases the stacking of specific single-stranded bases in 5 S RNA and aligns them in helical arrays, resulting in a conformation which facilitates base-pairing with nucleotide segment(s) of the ribosomal 23 S RNA or the transfer RNA (or both). The simple and precise difference CD method described here is potentially useful for studying subtle conformational changes of other nucleic acid-protein interactions.     

Base-stacking interactions in double-helical DNA structures: experiment versus theory

Atom-atom potential energy calculations have been undertaken for deriving stacking energies in double-helical structures. A comparison between the energy patterns of A- and B-type double-helical fragments determined by single-crystal X-ray diffraction methods versus idealized uniform models based on fibre diffraction data shows that the van der Waals stacking energy is largely sensitive to local changes in the relative orientation of adjacent base pairs. The sequence-dependent conformational variability observed in the high-resolution structures appears to be a consequence of the equipartitioning of the stacking energy along the double helix. The large energy variations expected for a uniform structure are dampened considerably in the observed structures by means of local changes in conformational features such as helix rotation and roll angles between base pairs.     

Conformational energy calculations for dinucleotide molecules. A systematic study of dinucleotide conformation, with application to diadenosine pyrophosphate.

A systematic study of the conformational states of the dinucleotide diadenosine 5′,5′-pyrophosphate (AppA), an analog of the coenzyme NAD⁺, has been made using semi-empirical energy calculations. Taking low-energy mononucleotide structures as starting conformations, energy minimizations have been performed. The most stable structures exhibit stacking interactions between the adenine bases; there are many different stacked states of similar energy; their stability is derived from nonbonded interactions primarily between the bases but also from base–sugar interactions. The most common form of stacking in the most stable structures was found to be antiparallel A-A helix. These findings are consistent with the experimental data, which suggest that AppA adopts predominantly a stacked state in solution, and this state incorporates a variety of stacked conformations.     

On loop folding in nucleic acid hairpin-type structures

In a series of studies, combining NMR, optical melting and T-jump experiments, it was found that DNA hairpins display a maximum stability when the loop part of the molecule comprises four or five nucleotide residues. This is in contrast with the current notion based on RNA hairpin studies, from which it had been established that a maximum hairpin stability is obtained for six or seven residues in the loop. Here we present a structural model to rationalize these observations. This model is based on the notion that to a major extent base stacking interactions determine the stability of nucleic acid conformations. The model predicts that loop folding in RNA is characterized by an extension of the base stacking at the 5'-side of the double helix by five or six bases; the remaining gap can then easily be closed by two nucleotides. Conversely, loop folding in DNA is characterized by extending base stacking at the 3'-side of the double helical stem by two or three residues; again bridging of the remaining gap can then be achieved by one or two nucleotides. As an example of loop folding in RNA the anticodon loop of yeast tRNAPhe is discussed. For the DNA hairpin formed by d(ATCCTAT4TAGGAT) it is shown that the loop structure obtained from molecular mechanics calculations obeys the above worded loop folding principles.     

Co-optimization of ribozyme substrate stacking and L-arginine binding.

A model of the Tetrahymena catalytic site predicts that nucleotide 262 (nt262) caps an RNA pocket in which nucleoside substrates and arginine-like competitive inhibitors reside. Here we show that substituted RNAs behave as if nt262 stacks on nucleoside substrates, supporting the model. The more frequent an nt262 is in natural sequences, the more reactive the corresponding Tetrahymena RNA is for both cognate and non-cognate nucleoside substrates. These more reactive RNAs with the majority nt262 also bind arginine more strongly, stereoselect more strongly in favor of L-arginine, and make a greater distinction between the somewhat similar side-chains of L-arginine and L-lysine. These parallel changes in interaction with nucleosides and arginine analogs seem best explained by stacking of the arginine's guanidino group under the nt262 base. One consequence is that selection for improved Tetrahymena catalysis with nucleosides should also yield an improved arginine site.     

Synthetic oligoribonucleotides carrying site-specific modifications for RNA structure-function analysis

Synthetic oligoribonucleotides have become increasingly valuable in studies of RNA structure and function. A range of nucleotide analogues is available which carry modifications in the base, sugar or phosphate moieties. Such analogues have been incorporated into synthetic RNA structures to eliminate or alter individual functional groups in the RNA which potentially can take part in hydrogen-bonding or other non-covalent interactions. Comparisons of the properties of the modified RNAs with unmodified RNA models allow conclusions to be drawn concerning the importance or otherwise of specific functional groups within the RNA. These methods have been applied to studies of RNA interactions with proteins, RNA catalysis and RNA structure.     

Synthesis and stereochemical characterization of diastereomeric nucleoside-phosphorothioselenoates by NMR methods

Synthesis and stereochemical characterization of enantiomerically pure nucleoside-phosphorothioselenoates are reported. The effects of solvent and temperature on the vicinal carbon-phosphorus couplings are described and the results are interpreted in terms of conformational changes influenced by stacking interactions between the bases and the phenyl rings.