2-Thiouracil deprived of thiocarbonyl function preferentially base pairs with guanine rather than adenine in RNA and DNA duplexes

2-Thiouracil-containing nucleosides are essential modified units of natural and synthetic nucleic acids. In particular, the 5-substituted-2-thiouridines (S2Us) present in tRNA play an important role in tuning the translation process through codon–anticodon interactions. The enhanced thermodynamic stability of S2U-containing RNA duplexes and the preferred S2U-A versus S2U-G base pairing are appreciated characteristics of S2U-modified molecular probes. Recently, we have demonstrated that 2-thiouridine (alone or within an RNA chain) is predominantly transformed under oxidative stress conditions to 4-pyrimidinone riboside (H2U) and not to uridine. Due to the important biological functions and various biotechnological applications for sulfur-containing nucleic acids, we compared the thermodynamic stabilities of duplexes containing desulfured products with those of 2-thiouracil-modified RNA and DNA duplexes. Differential scanning calorimetry experiments and theoretical calculations demonstrate that upon 2-thiouracil desulfuration to 4-pyrimidinone, the preferred base pairing of S2U with adenosine is lost, with preferred base pairing with guanosine observed instead. Therefore, biological processes and in vitro assays in which oxidative desulfuration of 2-thiouracil-containing components occurs may be altered. Moreover, we propose that the H2U-G base pair is a suitable model for investigation of the preferred recognition of 3′-G-ending versus A-ending codons by tRNA wobble nucleosides, which may adopt a 4-pyrimidinone-type structural motif.     

Crystal structure of Staphylococcus aureus tRNA adenosine deaminase TadA in complex with RNA

Bacterial tRNA adenosine deaminases (TadAs) catalyze the hydrolytic deamination of adenosine to inosine at the wobble position of tRNA(Arg2), a process that enables this single tRNA to recognize three different arginine codons in mRNA. In addition, inosine is also introduced at the wobble position of multiple eukaryotic tRNAs. The genes encoding these deaminases are essential in bacteria and yeast, demonstrating the importance of their biological activity. Here we report the crystallization and structure determination to 2.0 A of Staphylococcus aureus TadA bound to the anticodon stem-loop of tRNA(Arg2) bearing nebularine, a non-hydrolyzable adenosine analog, at the wobble position. The cocrystal structure reveals the basis for both sequence and structure specificity in the interactions of TadA with RNA, and it additionally provides insight into the active site architecture that promotes efficient hydrolytic deamination.     

An unwinding activity that covalently modifies its double-stranded RNA substrate

An activity that unwinds double-stranded RNA has been reported to exist in several organisms. We have analyzed the RNA intermediates and final products of the unwinding reaction. Although the RNA becomes sensitive to single strand-specific ribonucleases during the reaction, the duplex is never completely unwound. Furthermore, the base pairing properties of the RNA are permanently altered; the reacted RNA cannot rehybridize to form the original duplex. We demonstrate that during the reaction many, but not all, of the adenosine residues are converted to inosine residues, and we propose that the covalent modification is responsible for the irreversible change in base pairing properties. Possible biological roles for the unwinding/modifying activity, as well as its relevance to antisense RNA experiments, are discussed.     

Mutation in the D arm enables a suppressor with a CUA anticodon to read both amber and ochre codons in Escherichia coli

Su9 of Escherichia coli differs from tRNATrp by only a G to A transition in the D arm, yet has an enhanced ability to translate UGA by an unusual C X A wobble pairing. In order to examine the effects of this mutation on translation of the complementary and wobble codons in vivo, we constructed the gene for an amber (UAG) suppressing variant of Su9, trpT179, by making the additional nucleotide change required for an amber suppressor anticodon. The resultant suppressor tRNA, Su79, is a very strong amber suppressor. Furthermore, the D arm mutation enables Su79 to suppress ochre (UAA) codons by C X A wobble pairing. These data demonstrate that the effect of the D arm mutation on wobble pairing is not restricted to a CCA anticodon. The effect extends to the CUA anticodon of Su79, thereby creating a new type of ochre suppressor. The new coding activity of Su79 cannot be explained by alterations in the level of aminoacylation, steady-state tRNA concentration, or nucleotide modification. The A24 mutation could permit unorthodox wobble pairings by generally enhancing tRNA efficiency at all codons or by altering codon specificity.     

The Paradox of Dual Roles in the RNA World: Resolving the Conflict Between Stable Folding and Templating Ability

The hypothesized dual roles of RNA as both information carrier and biocatalyst during the earliest stages of life require a combination of features: good templating ability (for replication) and stable folding (for ribozymes). However, this poses the following paradox: well-folded sequences are poor templates for copying, but poorly folded sequences are unlikely to be good ribozymes. Here, we describe a strategy to overcome this dilemma through G:U wobble pairing in RNA. Unlike Watson–Crick base pairs, wobble pairs contribute highly to the energetic stability of the folded structure of their sequence, but only slightly, if at all, to the stability of the folded reverse complement. Sequences in the RNA World might thereby combine stable folding of the ribozyme with an unstructured, reverse-complementary genome, resulting in a “division of labor” between the strands. We demonstrate this strategy using computational simulations of RNA folding and an experimental model of early replication, nonenzymatic template-directed RNA primer extension. Additional study is needed to solve other problems associated with a complete replication cycle, including separation of strands after copying. Interestingly, viroid RNA sequences, which have been suggested to be relics of an RNA World (Diener, Proc Natl Acad Sci USA 86:9370–9374, 1989), also show significant asymmetry in folding energy between the infectious (+) and template (?) strands due to G:U pairing, suggesting that this strategy may even be used by replicators in the present day.     

The G x U wobble base pair. A fundamental building block of RNA structure crucial to RNA function in diverse biological systems

The G x U wobble base pair is a fundamental unit of RNA secondary structure that is present in nearly every class of RNA from organisms of all three phylogenetic domains. It has comparable thermodynamic stability to Watson-Crick base pairs and is nearly isomorphic to them. Therefore, it often substitutes for G x C or A x U base pairs. The G x U wobble base pair also has unique chemical, structural, dynamic and ligand-binding properties, which can only be partially mimicked by Watson-Crick base pairs or other mispairs. These features mark sites containing G x U pairs for recognition by proteins and other RNAs and allow the wobble pair to play essential functional roles in a remarkably wide range of biological processes.     

Drosophila ribosomal RNA genes function as an X-Y pairing site during male meiosis

In Drosophila melanogaster males, the sex chromosomes pair during meiosis in the centric X heterochromatin and at the base of the short arm of the Y (YS), in the vicinity of the nucleolus organizers. X chromosomes deficient for the pairing region segregate randomly from the Y. In this report we show that a single ribosomal RNA (rRNA) gene stimulates X-Y pairing and disjunction when inserted onto a heterochromatically deficient X chromosome by P element-mediated transformation. We also show that insert-containing X chromosomes pair at the site of insertion, that autosomal rDNA inserts do not affect X-Y pairing or disjunction, and that the strength of an X pairing site is proportional to the dose of ectopic rRNA genes. These results demonstrate that rRNA genes can promote X-Y pairing and disjunction and imply that the nucleolus organizers function as X-Y pairing sites in wild-type Drosophila males.     

Kinetics of formation of hypoxanthine containing base pairs by HIV-RT: RNA template effects on the base substitution frequencies

Hypoxanthine (H), the deamination product of adenine, has been implicated in the high frequency of A to G transitions observed in retroviral and other RNA genomes. Although H·C base pairs are thermodynamically more stable than other H·N pairs, polymerase selection may be determined in part by kinetic factors. Therefore, the hypoxanthine induced substitution pattern resulting from replication by viral polymerases may be more complex than that predicted from thermodynamics. We have examined the steady-state kinetics of formation of base pairs opposite template H in RNA by HIV-RT, and for the incorporation of dITP during first- and second-strand synthesis. Hypoxanthine in an RNA template enhances the k_2app for pairing with standard dNTPs by factors of 10–1000 relative to adenine at the same sequence position. The order of base pairing preferences for H in RNA was observed to be H·C >> H·T > H·A > H·G. Steady-state kinetics of insertion for all possible mispairs formed with dITP were examined on RNA and DNA templates of identical sequence. Insertion of dITP opposite all bases occurs 2–20 times more frequently on RNA templates. This bias for higher insertion frequencies on RNA relative to DNA templates is also observed for formation of mispairs at template A. This kinetic advantage afforded by RNA templates for mismatches and pairing involving H suggests a higher induction of mutations at adenines during first-strand synthesis by HIV-RT.     

Configuration of wobble base pairs having pyrimidines as anticodon wobble bases: significance for codon degeneracy

Degeneracy of the genetic code was attributed by Crick to imprecise hydrogen-bonded base-pairing at the wobble position during codon–anticodon pairing. The Crick wobble rules define but do not explain the RNA base pair combinations allowed at this position. We select six pyrimidine bases functioning as anticodon wobble bases (AWBs) to study their H-bonded pairing properties with the four major RNA bases using density functional theory at the B3LYP/6-31G(d,p) level. This is done to assess the extent to which the configuration of a solitary RNA wobble base pair may in itself determine specificity and degeneracy of the genetic code by allowing or disallowing the given base pair during codon–anticodon pairing. Calculated values of select configuration markers for the base pairs screen well between allowed and disallowed base pairs for most cases examined here, where the base pair width emerges as an important factor. A few allowed wobble pairs invoke the involvement of RNA nucleoside conformation, as well as involvement of the exocyclic substituent in H-bonding. This study, however, cannot explain the disallowed status of the Ura?Gua wobble pair on the basis of configuration alone. Explanation of the allowed status of the V?Ura pair requires further study on the mediatory role of water molecules. Apart from these two cases, these computational results are sufficient, on the basis of base pair configuration alone, to account for the specificity and degeneracy of the genetic code for all known cases of codon–anticodon pairing which involve the pyrimidine AWBs studied here.     

RNAi is antagonized by A→I hyper-editing

RNA interference (RNAi) and adenosine to inosine conversion are both mechanisms that respond to double-stranded RNA (dsRNA) and have been suggested to have antiviral roles. RNAi involves processing of dsRNA to short interfering RNAs (siRNAs), which subsequently mediate degradation of the cognate mRNAs. Deamination of adenosines changes the coding capacity of the RNA, as inosine is decoded as guanosine, and alters the structure because A–U base pairs are replaced by I•U wobble pairs. Here we show that RNAi is inhibited if the triggering dsRNA is first deaminated by ADAR2. Moreover, we show that production of siRNAs is progressively inhibited with increasing deamination and that this is sufficient to explain the inhibition of RNAi upon hyper-editing of dsRNAs.     

Decoding the genome: a modified view

Transfer RNA’s role in decoding the genome is critical to the accuracy and efficiency of protein synthesis. Though modified nucleosides were identified in RNA 50 years ago, only recently has their importance to tRNA’s ability to decode cognate and wobble codons become apparent. RNA modifications are ubiquitous. To date, some 100 different posttranslational modifications have been identified. Modifications of tRNA are the most extensively investigated; however, many other RNAs have modified nucleosides. The modifications that occur at the first, or wobble position, of tRNA’s anticodon and those 3′-adjacent to the anticodon are of particular interest. The tRNAs most affected by individual and combinations of modifications respond to codons in mixed codon boxes where distinction of the third codon base is important for discriminating between the correct cognate or wobble codons and the incorrect near-cognate codons (e.g. AAA/G for lysine versus AAU/C asparagine). In contrast, other modifications expand wobble codon recognition, such as U·U base pairing, for tRNAs that respond to multiple codons of a 4-fold degenerate codon box (e.g. GUU/A/C/G for valine). Whether restricting codon recognition, expanding wobble, enabling translocation, or maintaining the messenger RNA, reading frame modifications appear to reduce anticodon loop dynamics to that accepted by the ribosome. Therefore, we suggest that anticodon stem and loop domain nucleoside modifications allow a limited number of tRNAs to accurately and efficiently decode the 61 amino acid codons by selectively restricting some anticodon–codon interactions and expanding others.     

Deamination of mammalian glutamate receptor RNA by Xenopus dsRNA adenosine deaminase: similarities to in vivo RNA editing.

Double-stranded RNA (dsRNA) adenosine deaminase (dsRAD) converts adenosines to inosines within dsRNA. A great deal of evidence suggests that dsRAD or a related enzyme edits mammalian glutamate receptor mRNA in vivo. Here we map the deamination sites that occur in a truncated glutamate receptor-B (gluR-B) mRNA after incubation with pure Xenopus dsRAD. We find remarkable similarities, as well as distinct differences, between the observed deamination sites and the sites reported to be edited within RNAs isolated from mammalian brain. For example, although deamination at the biologically relevant Q/R editing site occurs, it occurs much less frequently than editing at this site in vivo. We hypothesize that the similarities between the deamination and editing patterns exist because the deamination specificity that is intrinsic to dsRAD is involved in selecting editing sites in vivo. We propose that the observed differences are due to the absence of accessory factors that play indirect roles in vivo, such as binding to and occluding certain sites from dsRAD, or promoting the RNA structure required for correct and efficient editing. The work reported here also suggests that dsRAD is capable of much more selectivity than previously thought; a minimal number of deamination sites (average < or = 5) were found in each gluR-B RNA. We speculate that the observed selectivity is due to the various structural elements (mismatches, bulges, loops) that periodically interrupt the base paired region required for editing.     

Evolution and RNA Relics. A Systems Biology View

The genetic code has evolved from its initial non-degenerate wobble version until reaching its present state of degeneracy. By using the stereochemical hypothesis, we revisit the problem of codon assignations to the synonymy classes of amino-acids. We obtain these classes with a simple classifier based on physico-chemical properties of nucleic bases, like hydrophobicity and molecular weight. Then we propose simple RNA (or more generally XNA, with X for D, P or R) ring structures that present, overlap included, one and only one codon by synonymy class as solutions of a combinatory variational problem. We compare these solutions to sequences of present RNAs considered as relics, with a high interspecific invariance, like invariant parts of (t)RNAs and micro-RNAs. We conclude by emphasizing some optimal properties of the genetic code.     

Structure and function of the conserved 690 hairpin in Escherichia coli 16 S ribosomal RNA: analysis of the stem nucleotides

Nucleotides 680 to 710 of Escherichia coli 16 S rRNA form a distinct structural domain required for ribosome function. The goal of this study was to determine the functional significance of pairing interactions in the 690 region. Two different secondary structures were proposed for this hairpin, based on phylogenetic and chemical modification studies. To study the effect of pairing interactions in the 690 hairpin on ribosome function and to determine which of the proposed secondary structures is biologically significant, we performed an instant-evolution experiment in which the nine nucleotides that form the proposed base-pairs and dangling ends of the 690 stem were randomly mutated, and functional mutant combinations were selected. A total of 96 unique functional mutants were isolated, assayed in vivo, and sequenced. Analysis of these data revealed extensive base-pairing and stacking interactions among the mutated nucleotides. Formation of either a Watson-Crick base-pair or G.U pair between positions 688 and 699 is absolutely required for ribosome function. We also performed NMR studies of a 31-nucleotide RNA which indicate the formation of a functionally important base-pair between nucleotides 688 and 699. Formation of a second base-pair between positions 689 and 698, however, is not essential for ribosome function, but the level of ribosome function correlates with the predicted thermodynamic stability of the nucleotide pairs in these positions. The universally conserved positions G690 and U697 are generally portrayed as forming a G.U mismatch. Our data show co-variation between these positions, but do not support the hypothesis that the G690:U697 pair forms a wobble structure. NMR studies of model 14-nt and 31-nt RNAs support these findings and show that G690 and U697 are involved in unusual stacking interactions but do not form a wobble pair. Preliminary NMR structural analysis reveals that the loop portion of the 690 hairpin folds into a highly structured and novel conformation.