Large telomerase RNA, telomere length heterogeneity and escape from senescence in Candida glabrata.

Telomerase, the key enzyme essential for the maintenance of eukaryotic chromosome ends, contains a reverse transcriptase and an RNA that provides the template for the synthesis of telomeric repeats. Here, we characterize the telomerase subunits in the hemiascomycete yeast Candida glabrata. We propose a secondary structure model for the telomerase RNA that is the largest described to date. Telomerase deletion mutants show a progressive shortening of telomeres and a modest loss of viability. Frequent post-senescence survivors emerge that possess long telomeric repeat tracts. We suggest that the high telomere length heterogeneity accounts for this distinct senescence phenotype.

The interaction networks of structured RNAs.

All pairwise interactions occurring between bases which could be detected in three-dimensional structures of crystallized RNA molecules are annotated on new planar diagrams. The diagrams attempt to map the underlying complex networks of base-base interactions and, especially, they aim at conveying key relationships between helical domains: co-axial stacking, bending and all Watson-Crick as well as non-Watson-Crick base pairs. Although such wiring diagrams cannot replace full stereographic images for correct spatial understanding and representation, they reveal structural similarities as well as the conserved patterns and distances between motifs which are present within the interaction networks of folded RNAs of similar or unrelated functions. Finally, the diagrams could help devising methods for meaningfully transforming RNA structures into graphs amenable to network analysis.

The A-minor motifs in the decoding recognition process.

The formation of A-minor motifs, mediated by adenines binding into the shallow/minor groove of stacked and helical Watson-Crick base pairs, is described. The conformations of the bacterial ribosomal decoding A site in various crystal structures are reviewed. The adenines A1492 and A1493 of the A site are seen either tucked in within the internal loop or bulging out and poised for interaction. This dynamic equilibrium contributes to the decoding process of the codon:anticodon base pairings. Aminoglycoside antibiotics lock the conformation of the A site in a single state with bulged-out adenines and thereby disrupt regulation of the decoding process.

A molecular dynamics simulation study of an aminoglycoside/A-site RNA complex: conformational and hydration patterns.

Aminoglycoside antibiotics interfere with the translation mechanism by binding to the tRNA decoding site of the 16S ribosomal RNA. Crystallographic structures of aminoglycosides bound to A-site systems clarified many static aspects of RNA-ligand interactions. To gain some insight on the dynamic aspects of recognition phenomena, we conducted molecular dynamics simulations of the aminoglycoside paromomycin bound to a eubacterial ribosomal decoding A-site oligonucleotide. Results from 25 ns of simulation time revealed that: (i) the neamine part of the antibiotic represents the main anchor for binding, (ii) additional sugar rings provide limited and fragile contacts, (iii) long-resident water molecules present at the drug/RNA interface are involved in the recognition phenomena. The combination of MD simulations together with systematic structural information offers striking insights into the molecular recognition processes underlying RNA/aminoglycoside binding. Important methodological considerations related to the use of medium resolution starting structures and associated sampling problems are thoroughly discussed.

Mutagenesis of 16S rRNA C1409-G1491 base-pair differentiates between 6'OH and 6'NH3+ aminoglycosides.

Using a single rRNA allelic Gram-positive model system, we systematically mutagenized 16S rRNA positions 1409 and 1491 to probe the functional relevance of structural interactions between aminoglycoside antibiotics and the A-site rRNA that were suggested by X-ray crystallography. At the structural level, the interaction of the 2-deoxystreptamine aminoglycosides with the rRNA base-pair C1409-G1491 has been suggested to involve the following features: (i) ring I of the disubstituted 2-deoxystreptamines stacks upon G1491 and H-bonds to the Watson-Crick edge of A1408; (ii) ring III of the 4,5-disubstituted aminoglycosides shows hydrogen bonding to G1491. However, we found that mutants with altered 16S rRNA bases 1409 and 1491 discriminated poorly between 4,5-disubstituted and 4,6-disubstituted 2-deoxystreptamines, but differentially affected aminoglycosides with a hydroxyl group versus an ammonium group at position 6' of ring I, e.g. G1491U conferred high-level drug resistance to paromomycin and geneticin, but not to neomycin, tobramycin or gentamicin.

Motif prediction in ribosomal RNAs Lessons and prospects for automated motif prediction in homologous RNA molecules.

The traditional way to infer RNA secondary structure involves an iterative process of alignment and evaluation of covariation statistics between all positions possibly involved in basepairing. Watson-Crick basepairs typically show covariations that score well when examples of two or more possible basepairs occur. This is not necessarily the case for non-Watson-Crick basepairing geometries. For example, for sheared (trans Hoogsteen/Sugar edge) pairs, one base is highly conserved (always A or mostly A with some C or U), while the other can vary (G or A and sometimes C and U as well). RNA motifs consist of ordered, stacked arrays of non-Watson-Crick basepairs that in the secondary structure representation form hairpin or internal loops, multi-stem junctions, and even pseudoknots. Although RNA motifs occur recurrently and contribute in a modular fashion to RNA architecture, it is usually not apparent which bases interact and whether it is by edge-to-edge H-bonding or solely by stacking interactions. Using a modular sequence-analysis approach, recurrent motifs related to the sarcin-ricin loop of 23S RNA and to loop E from 5S RNA were predicted in universally conserved regions of the large ribosomal RNAs (16S- and 23S-like) before the publication of high-resolution, atomic-level structures of representative examples of 16S and 23S rRNA molecules in their native contexts. This provides the opportunity to evaluate the predictive power of motif-level sequence analysis, with the goal of automating the process for predicting RNA motifs in genomic sequences. The process of inferring structure from sequence by constructing accurate alignments is a circular one. The crucial link that allows a productive iteration of motif modeling and realignment is the comparison of the sequence variations for each putative pair with the corresponding isostericity matrix to determine which basepairs are consistent both with the sequence and the geometrical data.

Group I introns and RNA folding.

Before the discovery of catalytic RNA, tRNA molecules were the most studied RNA molecules for understanding RNA folding. Afterwards, group I introns, because of their stability and the fact that structural folding could be monitored by following their catalytic activity, became the molecule of choice for studying RNA architecture and folding. A major advantage of group I introns for studying the catalytic activity of RNA molecules is that catalytic activity is triggered by the addition of external guanosine cofactors. The self-splicing activity can therefore be precisely controlled. Using group I introns, several RNA motifs central to RNA-RNA self-assembly and folding were discovered. The analysis of the recent X-ray structures of the rRNA subunits indicates that several motifs present in the ribosome occur also in various group I introns.

Crystal structure of paromomycin docked into the eubacterial ribosomal decoding A site.

BACKGROUND: Aminoglycoside antibiotics interfere with translation in both gram-positive and gram-negative bacteria by binding to the tRNA decoding A site of the 16S ribosomal RNA. RESULTS: Crystals of complexes between oligoribonucleotides incorporating the sequence of the ribosomal A site of Escherichia coli and the aminoglycoside paromomycin have been solved at 2.5 A resolution. Each RNA fragment contains two A sites inserted between Watson-Crick pairs. The paromomycin molecules interact in an enlarged deep groove created by two bulging and one unpaired adenines. In both sites, hydroxyl and ammonium side chains of the antibiotic form 13 direct hydrogen bonds to bases and backbone atoms of the A site. In the best-defined site, 8 water molecules mediate 12 other hydrogen bonds between the RNA and the antibiotics. Ring I of paromomycin stacks over base G1491 and forms pseudo-Watson-Crick contacts with A1408. Both the hydroxyl group and one ammonium group of ring II form direct and water-mediated hydrogen bonds to the U1495oU1406 pair. The bulging conformation of the two adenines A1492 and A1493 is stabilized by hydrogen bonds between phosphate oxygens and atoms of rings I and II. The hydrophilic sites of the bulging A1492 and A1493 contact the shallow groove of G=C pairs in a symmetrical complex. CONCLUSIONS: Water molecules participate in the binding specificity by exploiting the antibiotic hydration shell and the typical RNA water hydration patterns. The observed contacts rationalize the protection, mutation, and resistance data. The crystal packing mimics the intermolecular contacts induced by aminoglycoside binding in the ribosome.

Statistical analysis of atomic contacts at RNA-protein interfaces.

Forty-five crystals of complexes between proteins and RNA molecules from the Protein Data Bank have been statistically surveyed for the number of contacts between RNA components (phosphate, ribose and the four bases) and amino acid side chains. Three groups of complexes were defined: the tRNA synthetases; the ribosomal complexes; and a third group containing a variety of complexes. The types of atomic contacts were a priori classified into ionic, neutral H-bond, C-H...O H-bond, or van der Waals interaction. All the contacts were organized into a relational database which allows for statistical analysis. The main conclusions are the following: (i) in all three groups of complexes, the most preferred amino acids (Arg, Asn, Ser, Lys) and the less preferred ones (Ala, Ile, Leu, Val) are the same; Trp and Cys are rarely observed (respectively 15 and 5 amino acids in the ensemble of interfaces); (ii) of the total number of amino acids located at the interfaces 22% are hydrophobic, 40% charged (positive 32%, negative 8%), 30% polar and 8% are Gly; (iii) in ribosomal complexes, phosphate is preferred over ribose, which is preferred over the bases, but there is no significant preference in the other two groups; (iv) there is no significant prevalence of a base type at protein-RNA interfaces, but specifically Arg and Lys display a preference for phosphate over ribose and bases; Pro and Asn prefer bases over ribose and phosphate; Met, Phe and Tyr prefer ribose over phosphate and bases. Further, Ile, Pro, Ser prefer A over the others; Leu prefers C; Asp and Gly prefer G; and Asn prefers U. Considering the contact types, the following conclusions could be drawn: (i) 23% of the contacts are via potential H-bonds (including CH...O H-bonds and ionic interactions), 72% belong to van der Waals interactions and 5% are considered as short contacts; (ii) of all potential H-bonds, 54% are standard, 33% are of the C-H...O type and 13% are ionic; (iii) the Watson-Crick sites of G, O6(G) and principally N2(G) and the hydroxyl group O2' is more often involved in H-bonds than expected; the protein main chain is involved in 32% and the side chains in 68% of the H-bonds; considering the neutral and ionic H-bonds, the following couples are more frequent than expected-base A-Ser, base G-Asp/Glu, base U-Asn. The RNA CH groups interact preferentially with oxygen atoms (62% on the main chain and 19% on the side chains); (iv) the bases are involved in 38% of all H-bonds and more than 26% of the H-bonds have the H donor group on the RNA; (v) the atom O2' is involved in 21% of all H-bonds, a number greater than expected; (vi) amino acids less frequently in direct contact with RNA components interact frequently via their main chain atoms through water molecules with RNA atoms; in contrast, those frequently observed in direct contact, except Ser, use instead their side chain atoms for water bridging interactions.

Bulged residues promote the progression of a loop-loop interaction to a stable and inhibitory antisense-target RNA complex.

In several groups of bacterial plasmids, antisense RNAs regulate copy number through inhibition of replication initiator protein synthesis. These RNAs are characterized by a long hairpin structure interrupted by several unpaired residues or bulged loops. In plasmid R1, the inhibitory complex between the antisense RNA (CopA) and its target mRNA (CopT) is characterized by a four-way junction structure and a side-by-side helical alignment. This topology facilitates the formation of a stabilizer intermolecular helix between distal regions of both RNAs, essential for in vivo control. The bulged residues in CopA/CopT were shown to be required for high in vitro binding rate and in vivo activity. This study addresses the question of why removal of bulged nucleotides blocks stable complex formation. Structure mapping, modification interference, and molecular modeling of bulged-less mutant CopA-CopT complexes suggests that, subsequent to loop-loop contact, helix propagation is prevented. Instead, a fully base paired loop-loop interaction is formed, inducing a continuous stacking of three helices. Consequently, the stabilizer helix cannot be formed, and stable complex formation is blocked. In contrast to the four-way junction topology, the loop-loop interaction alone failed to prevent ribosome binding at its loading site and, thus, inhibition of RepA translation was alleviated.

Protein-dependent transition states for ribonucleoprotein assembly.

Native folding and splicing by the Saccharomyces cerevisiae mitochondrial bI5 group I intron RNA is facilitated by both the S. cerevisiae CBP2 and Neurospora crassa CYT-18 protein cofactors. Both protein-bI5 RNA complexes splice at similar rates, suggesting that the RNA active site structure is similar in both ribonucleoproteins. In contrast, the two proteins assemble with the bI5 RNA by distinct mechanisms and bind opposing, but partially overlapping, sides of the group I intron catalytic core. Assembly with CBP2 is limited by a slow, unimolecular RNA folding step characterized by a negligible activation enthalpy. We show that assembly with CYT-18 shows four distinctive features. (1) CYT-18 binds stably to the bI5 RNA at the diffusion controlled limit, but assembly to a catalytically active RNA structure is still limited by RNA folding, as visualized directly using time-resolved footprinting. (2) This mechanism of rapid stable protein binding followed by subsequent assembly steps has a distinctive kinetic signature: the apparent ratio of k(off) to k(on), determined in a partitioning experiment, differs from the equilibrium K(d) by a large factor. (3) Assembly with CYT-18 is characterized by a large activation enthalpy, consistent with a rate limiting conformational rearrangement. (4) Because assembly from the kinetically trapped state is faster at elevated temperature, we can identify conditions where CYT-18 accelerates (catalyzes) bI5 RNA folding relative to assembly with CBP2.

Four-way junctions in antisense RNA-mRNA complexes involved in plasmid replication control: a common theme?

In several groups of bacterial plasmids, antisense RNAs regulate copy number through inhibition of replication initiator protein synthesis. In plasmid R1, we have recently shown that the inhibitory complex between the antisense RNA (CopA) and its target mRNA (CopT) is characterized by the formation of two intermolecular helices, resulting in a four-way junction structure and a side-by-side helical alignment. Based on lead-induced cleavage and ribonuclease (RNase) V(1) probing combined with molecular modeling, a strikingly similar topology is supported for the complex formed between the antisense RNA (Inc) and mRNA (RepZ) of plasmid Col1b-P9. In particular, the position of the four-way junction and the location of divalent ion-binding site(s) indicate that the structural features of these two complexes are essentially the same in spite of sequence differences. Comparisons of several target and antisense RNAs in other plasmids further indicate that similar binding pathways are used to form the inhibitory antisense-target RNA complexes. Thus, in all these systems, the structural features of both antisense and target RNAs determine the topologically possible and kinetically favored pathway that is essential for efficient in vivo control.

Geometric nomenclature and classification of RNA base pairs.

Non-Watson-Crick base pairs mediate specific interactions responsible for RNA-RNA self-assembly and RNA-protein recognition. An unambiguous and descriptive nomenclature with well-defined and nonoverlapping parameters is needed to communicate concisely structural information about RNA base pairs. The definitions should reflect underlying molecular structures and interactions and, thus, facilitate automated annotation, classification, and comparison of new RNA structures. We propose a classification based on the observation that the planar edge-to-edge, hydrogen-bonding interactions between RNA bases involve one of three distinct edges: the Watson-Crick edge, the Hoogsteen edge, and the Sugar edge (which includes the 2'-OH and which has also been referred to as the Shallow-groove edge). Bases can interact in either of two orientations with respect to the glycosidic bonds, cis or trans relative to the hydrogen bonds. This gives rise to 12 basic geometric types with at least two H bonds connecting the bases. For each geometric type, the relative orientations of the strands can be easily deduced. High-resolution examples of 11 of the 12 geometries are presently available. Bifurcated pairs, in which a single exocyclic carbonyl or amino group of one base directly contacts the edge of a second base, and water-inserted pairs, in which single functional groups on each base interact directly, are intermediate between two of the standard geometries. The nomenclature facilitates the recognition of isosteric relationships among base pairs within each geometry, and thus facilitates the recognition of recurrent three-dimensional motifs from comparison of homologous sequences. Graphical conventions are proposed for displaying non-Watson-Crick interactions on a secondary structure diagram. The utility of the classification in homology modeling of RNA tertiary motifs is illustrated.

An extended structural signature for the tRNA anticodon loop.

Anticodon hairpins are structural motifs with contradictory functions. The recognition by aminoacyl synthetases implies extended interactions with the anticodon base triplet and thus, usually, an unfolding of the anticodon loop. The recognition by the ribosome and cognate interaction with a mRNA codon implies, on the other hand, the formation of a mini-helix with a canonical anticodon hairpin structure as observed by crystallography and NMR. To be able to understand the various properties of this motif, a precise description of its structural conservation is required. Here, on the basis of phylogenetic, structural, and molecular dynamics data, we discuss a conserved interaction established between the ribose of the U33 and the base at position 35, either a purine or a pyrimidine. This interaction involves the hydrogen bonding donor or acceptor potential of the hydroxyl group of U33 and has to be integrated in an extended definition of the anticodon hairpin. The extended structural signature provides also an explanation for the role played by pseudouridines at position 35.

Phylogenetic analysis of tmRNA genes within a bacterial subgroup reveals a specific structural signature.

Bacterial tmRNA mediates a trans-translation reaction, which permits the recycling of stalled ribosomes and probably also contributes to the regulated expression of a subset of genes. Its action results in the addition of a small number of C-terminal amino acids to protein whose synthesis had stalled and these constitute a proteolytic recognition tag for the degradation of these incompletely synthesized proteins. Previous work has identified pseudoknots and stem-loops that are widely conserved in divergent bacteria. In the present work an alignment of tmRNA gene sequences within 13 beta-proteobacteria reveals an additional sub-structure specific for this bacterial group. This sub-structure is in pseudoknot Pk2, and consists of one to two additional stem-loop(s) capped by stable GNRA tetraloop(s). Three-dimensional models of tmRNA pseudoknot 2 (Pk2) containing various topological versions of the additional sub-structure suggest that the sub-structures likely point away from the core of the RNA, containing both the tRNA and the mRNA domains. A putative tertiary interaction has also been identified.

Somatic mutation in human T-cell leukemia virus type 1 provirus and flanking cellular sequences during clonal expansion in vivo.

BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia/lymphoma, shows intrapatient genetic variability. Although HTLV-1 can replicate via the reverse transcription of virion RNA to a double-stranded DNA provirus (the conventional manner for retroviruses), its predominant mode of replication is via the clonal expansion (mitosis) of the infected cell. This expansion is achieved by the viral oncoprotein Tax, which keeps the infected CD4 T lymphocyte cycling. Because Tax also interferes with cellular DNA repair pathways, we investigated whether somatic mutations of the provirus that occur during the division of infected cells could account for HTLV-1 genetic variability. METHODS: An inverse polymerase chain reaction strategy was designed to distinguish somatic mutations from reverse transcription-associated substitutions. This strategy allows the proviral sequences to be isolated together with flanking cellular sequences. Using this method, we sequenced 208 HTLV-1 provirus 3' segments, together with their integration sites, belonging to 29 distinct circulating cellular clones from infected individuals. RESULTS: For 60% of the clones, 8%-80% of infected cells harbored a mutated HTLV-1 provirus, without evidence of reverse transcription-associated mutations. Mutations within flanking cellular sequences were also identified at a frequency of 2.8 x 10(-4) substitution per base pair. Some of these clones carried multiple discrete substitutions or deletions, indicating progressive accumulation of mutations during clonal expansion. The overall frequency of somatic mutations increased with the degree of proliferation of infected T cells. CONCLUSIONS: These data indicate that, in vivo, HTLV-1 variation results mainly from postintegration events that consist of somatic mutations of the proviral sequence occurring during clonal expansion. The finding of substitutions in flanking sequences suggests that somatic mutations occurring after integration, presumably coupled with selection, help move the cellular clones toward a transformed phenotype, of which adult T-cell leukemia/lymphoma is the end point.

Water and ion binding around r(UpA)12 and d(TpA)12 oligomers--comparison with RNA and DNA (CpG)12 duplexes.

The structural and dynamic properties of the water and ion first coordination shell of the r(A-U) and d(A-T) base-pairs embedded within the r(UpA)12 and d(TpA)12 duplexes are described on the basis of two 2.4 ns molecular dynamics simulations performed in a neutralizing aqueous environment with 0.25 M added KCl. The results are compared to previous molecular dynamics simulations of the r(CpG)12 and d(CpG)12 structures performed under similar conditions. It can be concluded that: (i) RNA helices are more rigid than DNA helices of identical sequence, as reflected by the fact that RNA duplexes keep their initial A-form shape while DNA duplexes adopt more sequence-specific shapes. (ii) Around these base-pairs, the water molecules occupy 21 to 22 well-defined hydration sites, some of which are partially occupied by potassium ions. (iii) These hydration sites are occupied by an average of 21.9, 21.0, 20.1, and 19.8 solvent molecules (water and ions) around the r(G=C), r(A-U), d(G=C), and d(A-T) pairs, respectively. (iv) From a dynamic point of view, the stability of the hydration shell is the strongest for the r(G=C) pairs and the weakest for the d(A-T) pairs. (v) For RNA, the observed long-lived hydration patterns are essentially non-sequence dependent and involve water bridges located in the deep groove and linking OR atoms of adjacent phosphate groups. Maximum lifetimes are close to 400 ps. (vi) In contrast, for DNA, long-lived hydration patterns are sequence dependent and located in the minor groove. For d(CpG)12, water bridges linking the (G)N3 and (C)O2 with the O4' atoms of adjacent nucleotides with 400 ps maximum lifetimes are characterized while no such bridges are observed for d(TpA)12. (vii) Potassium ions are observed to bind preferentially to deep/major groove atoms at RpY steps, essentially d(GpC), r(GpC), and r(ApU), by forming ion-bridges between electronegative atoms of adjacent base-pairs. On average, about half an ion is observed per base-pair. Positive ion-binding determinants are related to the proximity of two or more electronegative atoms. Negative binding determinants are associated with the electrostatic and steric hindrance due to the proximity of electropositive amino groups and neutral methyl groups. Potassium ions form only transient contacts with phosphate groups.

TectoRNA: modular assembly units for the construction of RNA nano-objects.

Structural information on complex biological RNA molecules can be exploited to design tectoRNAs or artificial modular RNA units that can self-assemble through tertiary interactions thereby forming nanoscale RNA objects. The selective interactions of hairpin tetraloops with their receptors can be used to mediate tectoRNA assembly. Here we report on the modulation of the specificity and the strength of tectoRNA assembly (in the nanomolar to micromolar range) by variation of the length of the RNA subunits, the nature of their interacting motifs and the degree of flexibility of linker regions incorporated into the molecules. The association is also dependent on the concentration of magnesium. Monitoring of tectoRNA assembly by lead(II) cleavage protection indicates that some degree of structural flexibility is required for optimal binding. With tectoRNAs one can compare the binding affinities of different tertiary motifs and quantify the strength of individual interactions. Furthermore, in analogy to the synthons used in organic chemistry to synthesize more complex organic compounds, tectoRNAs form the basic assembly units for constructing complex RNA structures on the nanometer scale. Thus, tectoRNA provides a means for constructing molecular scaffoldings that organize functional modules in three-dimensional space for a wide range of applications.

A potential RNA drug target in the hepatitis C virus internal ribosomal entry site.

Subdomain IlId from the hepatitis C virus (HCV) internal ribosome entry site (IRES) has been shown to be essential for cap-independent translation. We have conducted a structural study of a 27-nt fragment, identical in sequence to IlId, to explore the structural features of this subdomain. The proposed secondary structure of IlId is comprised of two 3 bp helical regions separated by an internal loop and closed at one end by a 6-nt terminal loop. NMR and molecular modeling were used interactively to formulate a validated model of the three-dimensional structure of IlId. We found that this fragment contains several noncanonical structural motifs and non-Watson-Crick base pairs, some of which are common to other RNAs. In particular, a motif characteristic of the rRNA alpha-sarcin/ricin loop was located in the internal loop. The terminal loop, 5'-UUGGGU, was found to fold to form a trinucleotide loop closed by a trans-wobble U.G base pair. The sixth nucleotide was bulged out to allow stacking of this U.G pair on the adjacent helical region. In vivo mutational analysis in the context of the full IRES confirmed the importance of each structural motif within IIId for IRES function. These findings may provide clues as to host cellular proteins that play a role in IRES-directed translation and, in particular, the mechanism through which host ribosomes are sequestered for viral function.