Divalent metal ions are essential for the folding and catalytic activities of many RNAs. A commonly employed biochemical technique to identify metal-binding sites in RNA is the rescue of RP α-phosphorothioate (PS) interference by the addition of soft divalent metal ions. To access the ability of such experiments to accurately identify metal-ion coordinations within a complex RNA fold, we report metal-rescue results from the Tetrahymena group I intron P4–P6 domain, where the location and coordination of five divalent metal ions have been determined by X-ray crystallography [J.H. Cate et al., Nat Struct Biol, 1997, 4:553]. We used a native gel mobility-shift to assay for P4–P6 folding in the presence of various divalent metal ions, and found that even moderate concentrations of Mn2+ (≥0.5 mM) can rescue PS interference at sites that do not coordinate metal ions within the P4–P6 crystal structure. To control for such effects, 2′-deoxynucleotide interference was used to titrate the Mn2+ concentration to a level that produces metal-ion-specific rescue (0.3 mM). This concentration of Mn2+ specifically rescued four of the six metal-dependent phosphorothioate effects within the RNA domain, including PS interference resulting from outer-sphere coordination to the metals. Both sites that were not specifically rescued make inner-sphere metal-ion coordinations. Cd2+ and Zn2+ afforded rescue at a smaller subset of the six metal-specific PS sites, though again phosphates making outer-sphere coordinations to metal ions were rescued preferentially. These data on P4–P6 domain folding reinforce the need for caution when interpreting metal-rescue experiments.

Programmed cell death by hok/sok of plasmid R1 and pnd/pndB of R483 mediates plasmid maintenance by killing of plasmid-free cells. It has been previously suggested that premature translation of the plasmid-mediated toxin is prevented during transcription of the hok and pnd mRNAs by the formation of metastable hairpins in the mRNA at the 5′ end. Here, experimental evidence is presented for the existence of metastable structures in the 5′ leader of the hok and pnd mRNAs in vitro. The kinetics of refolding from the metastable to the stable structure in the isolated fragments of the 5′ ends of both the hok and pnd mRNAs could be estimated, in agreement with the structural rearrangement in this region, as predicted to occur during transcription and mRNA activation. The refolding rates of hok and pnd structures are slow enough to allow for the formation of downstream hairpin structures during elongation of the mRNAs, which thereby helps to stabilize the metastable structures. Thus, the kinetic refolding parameters of the hok and pnd mRNAs are consistent with the proposal that the metastable structures prevent premature translation and/or antisense RNA binding during transcription.

The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein required for cotranslational targeting of proteins to the membrane of the endoplasmic reticulum of the bacterial plasma membrane. Domain IV of SRP RNA consists of a short stem-loop structure with two internal loops that contain the most conserved nucleotides of the molecule. All known essential interactions of SRP occur in that moiety containing domain IV. The solution structure of a 43-nt RNA comprising the complete Escherichia coli domain IV was determined by multidimensional NMR and restrained molecular dynamics refinement. Our data confirm the previously determined rigid structure of a smaller subfragment containing the most conserved, symmetric internal loop A (Schmitz et al., Nat Struct Biol, 1999, 6:634–638), where all conserved nucleotides are involved in nucleotide-specific structural interactions. Asymmetric internal loop B provides a hinge in the RNA molecule; it is partially flexible, yet also uniquely structured. The longer strand of internal loop B extends the major groove by creating a ledge-like arrangement; for loop B however, there is no obvious structural role for the conserved nucleotides. The structure of domain IV suggests that loop A is the initial site for the RNA/protein interaction creating specificity, whereas loop B provides a secondary interaction site.

Comparative sequence analysis complements experimental methods for the determination of RNA three-dimensional structure. This approach is based on the concept that different sequences within the same gene family form similar higher-order structures. The large number of rRNA sequences with sufficient variation, along with improved covariation algorithms, are providing us with the opportunity to identify new base triples in 16S rRNA. The three-dimensional conformations for one of our strongest candidates involving U121 (C124:G237) and/or U121 (U125:A236) (Escherichia coli sequence and numbering) are analyzed here with different molecular modeling tools. Molecular modeling shows that U121 interacts with C124 in the U121 (C124:G237) base triple. This arrangement maintains isomorphic structures for the three most frequent sequence motifs (approximately 93% of known bacterial and archaeal sequences), is consistent with chemical reactivity of U121 in E. coli ribosomes, and is geometrically favorable. Further, the restricted set of observed canonical (GU, AU, GC) base-pair types at positions 124:237 and 125:236 is consistent with the fact that the canonical base-pair sets (for both base pairs) that are not observed in nature prevent the formation of the 121(124:237) base triple. The analysis described here serves as a general scheme for the prediction of specific secondary and tertiary structure base pairing where there is a network of correlated base changes.

In Drosophila, the spliceosomal protein SNF fulfills the functions of two vertebrate proteins, U1 snRNP-U1A and U2 snRNP-U2B″. The structure and sequence of SNF, U1A, and U2B″ are nearly identical with two RNA recognition motifs (RRM) separated by a short linker region, yet they have different RNA-binding properties: U1A binds U1 snRNA, U2B″ binds U2 snRNA, and SNF binds both snRNAs. Structure/function studies on the human proteins have identified motifs in the N-terminal RRM that are critical for RNA-binding specificity but have failed to identify a function for the C-terminal RRM. Interestingly, SNF is chimeric in these motifs, suggesting a basis for its dual specificity. Here, we test the importance of these motifs by introducing site-directed mutations in the snf coding region and examining the effects of these mutations on assembly into the snRNP and on snf function in vivo. We found that an N-terminal RRM mutant protein predicted to eliminate RNA binding still assembles into snRNPs and is capable of rescuing snf 's lethal phenotype only if the normally dispensable C-terminal RRM is present. We also found that the mixed motif in the “RNA-specificity” domain is necessary for SNF's dual function whereas the mixed motif in the U2A′-protein-binding region is not. Finally, we demonstrate that animals carrying a snf mutation that converts SNF from a bifunctional protein to a U1 snRNP-specific protein are viable. This unexpected result suggests that SNF's presence within the U2 snRNP is not essential for splicing.

Certain viruses, transposons, and cellular genes have evolved specific sequences that induce high levels of specific translational errors. Such “programmed misreading” can result in levels of frameshifting or nonsense codon readthrough that are up to 1,000-fold higher than normal. Here we determine how a number of mutations in yeast affect the programmed misreading used by the yeast Ty retrotransposons. These mutations have previously been shown to affect the general accuracy of translational termination. We find that among four nonsense suppressor ribosomal mutations tested, one (a ribosomal protein mutation) enhanced the efficiency of the Ty1 frameshifting, another (an rRNA mutation) reduced frameshifting, and two others (another ribosomal protein mutation and another rRNA mutation) had no effect. Three antisuppressor rRNA mutations all reduced Ty1 frameshifting; however the antisuppressor mutation in the ribosomal protein did not show any effect. Among nonribosomal mutations, the allosuppressor protein phosphatase mutation enhanced Ty1 frameshifting, whereas the partially inactive prion form of the release factor eRF3 caused a slight decrease, if any effect. A mutant form of the other release factor, eRF1, also had no effect on frameshifting. Our data suggest that Ty frameshifting is under the control of the cellular translational machinery. Surprisingly we find that translational suppressors can affect Ty frameshifting in either direction, whereas antisuppressors have either no effect or cause a decrease.

A computer program, OligoWalk, is reported that predicts the equilibrium affinity of complementary DNA or RNA oligonucleotides to an RNA target. This program considers the predicted stability of the oligonucleotide-target helix and the competition with predicted secondary structure of both the target and the oligonucleotide. Both unimolecular and bimolecular oligonucleotide self structure are considered with a user-defined concentration. The application of OligoWalk is illustrated with three comparisons to experimental results drawn from the literature.

Through a combination of in vitro snRNP reconstitution, photocross-linking and immunoprecipitation techniques, we have investigated the interaction of proteins with the spliceosomal U6 snRNA in U6 snRNPs, U4/U6 di-snRNPs and U4/U6.U5 tri-snRNPs. Of the seven Lsm (Sm-like) proteins that associate specifically with this spliceosomal snRNA, three were shown to contact the RNA directly, and to maintain contact as the U6 RNA is incorporated into tri-snRNPs. In tri-snRNPs, the U5 snRNP protein Prp8 contacts position 54 of U6, which is in the conserved region that contributes to the formation of the catalytic core of the spliceosome. Other tri-snRNP-specific contacts were also detected, indicating the dynamic nature of protein interactions with this important snRNA. The uridine-rich extreme 3′ end of U6 RNA was shown to be essential but not sufficient for the association of the Lsm proteins. Interestingly, the Lsm proteins associate efficiently with the 3′ half of U6, which contains the 3′ stem-loop and uridine-rich 3′ end, suggesting that the Lsm and Sm proteins may recognize similar features in RNAs.

A 29-nt RNA catalyst successively forms the aminoacyl ester phe-RNA, and then peptidyl-RNA (phe-phe-RNA), given phenylalanine adenylate (phe-AMP) as substrate. Catalysis of two related reactions at similar rates supports the argument that RNA catalysts would evolve as groups with similar mechanisms. In particular, successive aminoacyl- and peptidyl-RNA synthesis by one RNA suggests that uncoded but RNA-catalyzed peptide synthesis would evolve before the synthesis of coded peptides.

The G[bull ]U pair at the third position in the acceptor helix of Escherichia coli tRNAAla is critical for aminoacylation. The features that allow G[bull ]U recognition are likely to include direct interaction of alanyl-tRNA synthetase with distinctive atomic groups and indirect recognition of the structural and stability information encoded in the sequence of G[bull ]U and its immediate context. The present work investigates the thermodynamic stability and acceptor activity for a comprehensive set of variant RNAs with substitutions of the G[bull ]U pair of E. coli tRNAAla. The four RNAs with Watson–Crick substitutions had a lower acceptor activity and a higher stability relative to the G[bull ]U RNA. On the other hand, the RNAs with mispair substitutions had a lower stability, but either a higher or a lower acceptor activity. Thus, the entire set of variant RNAs does not exhibit a correlation between thermodynamic stability of the free, unbound tRNA and its acceptor activity. The substantial acceptor activity of tRNAs with particular mispair substitutions may be explained by their ability to assume the conformational preferences of alanyl–tRNA synthetase. Moreover, the G[bull ]U pair may provide a point of deformability for the substrate tRNA to adapt to the enzyme's active site.

Two forms of spliceosomes were found in higher eukaryotes. The major form contains the U1, U2, U4, U5, and U6 snRNAs; the minor form contains the U11, U12, U4atac, U5, and U6atac snRNAs. Assembly and function of the major form are based on a complex dynamic of UsnRNA–UsnRNA and UsnRNA–pre-mRNA interactions, and the involved UsnRNA segments are highly posttranscriptionally modified in plants and vertebrates. To further characterize the minor form of spliceosomes, we looked for the Ψ residues in HeLa cells' U11, U12, U4atac, and U6atac snRNAs, using chemical approaches. Four Ψ residues were detected in total for these four atac UsnRNAs, compared to 20 in their counterparts of the major spliceosomes. The two Ψ residues detected in U12 are also found in U2 snRNA. One of them belongs to the branch-site-recognition sequence. It forms one of the base pairs that bulge out the A residue, responsible for the nucleophilic attack. Conservation of this strategic Ψ residue probably reflects a functional role. Another Ψ residue was detected in a U4atac snRNA segment involved in formation of helix II with U6atac. The fourth one was detected in the additional stem-loop structure present at the 3′ end of U6atac snRNA. Differences in Ψ content of the atac and major UsnRNAs of human cells may participate in the differentiation of the two splicing systems. Based on secondary structure similarity, U2 and U12 snRNAs on the one hand and U4 and U4atac snRNAs on the other hand may share common Ψ synthases.

A great deal of progress in elucidating the mechanisms of spliceosome assembly has been achieved by analyzing the A, B, and C spliceosomal complexes on native polyacrylamide gels. In contrast, progress in understanding the earliest spliceosomal complex E has been hampered because this complex dissociates on native gels and is difficult to detect by other methods. Here we report conditions for resolving the spliceosomal complex E using a native horizontal agarose mini-gel system. This system also provides a simple alternative to polyacrylamide gels for resolving the ATP-dependent spliceosomal complexes.

We describe a fast and simple one-step affinity-purification method for the isolation of specific RNA-binding proteins. An in vitro-transcribed hybrid RNA consisting of an aptamer sequence with high binding specificity to the antibiotic streptomycin and a putative protein-binding RNA sequence is incubated with crude extract. After complex formation, the sample is applied to an affinity column containing streptomycin immobilized to Sepharose. The binding of the in vitro-assembled RNA–protein complex to streptomycin-Sepharose is mediated by the aptamer RNA and the specifically bound proteins are recovered from the affinity matrix by elution with the antibiotic. Employing two well-characterized RNA–protein interactions, we tested the performance of this new method. The spliceosomal U1A protein and the bacteriophage MS2 coat protein could be isolated via their appropriate RNA motif containing hybrid RNA from crude yeast extracts in high yield and purity after only one round of affinity purification. As the purification principle is independent of the extract source, this new affinity chromatography strategy that makes use of an in vitro-selected antibiotic-binding RNA as a tag, “StreptoTag,” should be applicable to extracts from other organisms as well. Therefore, we propose StreptoTag to be a versatile tool for the isolation of unknown RNA-binding proteins.