RNA structural dynamics: pre-melting and melting transitions in E. coli 5S rRNA

The temperature dependent transition from duplex to a single strand in E. coli 5S ribosomal RNA is a multistep process, and it involves intermediate states. We have analyzed these structural dynamics by chemical modification of cytidines and by single strand specific nuclease digestions. This combined approach led to the characterization of premelting and melting transitions within individual structural segments of the native macromolecule, which we feel may find general application to the structure of biological polyribonucleotides: 1) G-C base pairs at the termini of helices are relatively unstable and they readily undergo premelting transition. 2) Internal G-U/A-U rich stretches of helices exhibit dynamic premelting properties. 3) Hairpin loops have a relatively stronger destabilizing effect than internal loops. 4) Bulge loops destabilize the neighbouring base pairs. 5) Melting of helical segments occurs starting from the destabilizing structures listed above, preferentially from the helix termini. E. coli 5S rRNA has been shown to adopt different conformations. The presence of urea leads to induction of enhancement in the sensitivity for nuclease S1 at several nucleotide positions. The possibility of structural rearrangements will be discussed.     

Symmetric K+ and Mg2+ ion-binding sites in the 5S rRNA loop E inferred from molecular dynamics simulations

Potassium binding to the 5 S rRNA loop E motif has been studied by molecular dynamics at high (1.0 M) and low (0.2 M) concentration of added KCl in the presence and absence of Mg²⁺. A clear pattern of seven deep groove K⁺ binding sites or regions, in all cases connected with guanine N7/O6 atoms belonging to GpG, GpA, and GpU steps, was identified, indicating that the LE deep groove is significantly more ionophilic than the equivalent groove of regular RNA duplexes. Among all, two symmetry-related sites (with respect to the central G·A pair) were found to accommodate K⁺ ions with particularly long residence times. In a preceding molecular dynamics study by Auffinger et al. in the year 2003, these two sites were described as constituting important Mg²⁺ binding locations. Altogether, the data suggest that these symmetric sites correspond to the loop E main ion binding regions. Indeed, they are located in the deep groove of an important ribosomal protein binding motif associated with a fragile pattern of non-Watson-Crick pairs that has certainly to be stabilized by specific Mg²⁺ ions in order to be efficiently recognized by the protein. Besides, the other sites accommodate monovalent ions in a more diffuse way pointing out their lesser significance for the structure and function of this motif. Ion binding to the shallow groove and backbone atoms was generally found to be of minor importance since, at the low concentration, no well defined binding site could be characterized while high K⁺ concentration promoted mostly unspecific potassium binding to the RNA backbone. In addition, several K⁺ binding sites were located in positions equivalent to water molecules from the first hydration shell of divalent ions in simulations performed with magnesium, indicating that ion binding regions are able to accommodate both mono- and divalent ionic species. Overall, the simulations provide a more precise but, at the same time, a more intricate view of the relations of this motif with its ionic surrounding.     

The 5S rRNA loop E: chemical probing and phylogenetic data versus crystal structure.

A significant fraction of the bases in a folded, structured RNA molecule participate in noncanonical base pairing interactions, often in the context of internal loops or multi-helix junction loops. The appearance of each new high-resolution RNA structure provides welcome data to guide efforts to understand and predict RNA 3D structure, especially when the RNA in question is a functionally conserved molecule. The recent publication of the crystal structure of the "Loop E" region of bacterial 5S ribosomal RNA is such an event [Correll CC, Freeborn B, Moore PB, Steitz TA, 1997, Cell 91:705-712]. In addition to providing more examples of already established noncanonical base pairs, such as purine-purine sheared pairings, trans-Hoogsteen UA, and GU wobble pairs, the structure provides the first high-resolution views of two new purine-purine pairings and a new GU pairing. The goal of the present analysis is to expand the capabilities of both chemical probing and phylogenetic analysis to predict with greater accuracy the structures of RNA molecules. First, in light of existing chemical probing data, we investigate what lessons could be learned regarding the interpretation of this widely used method of RNA structure probing. Then we analyze the 3D structure with reference to molecular phylogeny data (assuming conservation of function) to discover what alternative base pairings are geometrically compatible with the structure. The comparisons between previous modeling efforts and crystal structures show that the intricate involvements of ions and water molecules in the maintenance of non-Watson-Crick pairs render the process of correctly identifying the interacting sites in such pairs treacherous, except in cases of trans-Hoogsteen A/U or sheared A/G pairs for the adenine N1 site. The phylogenetic analysis identifies A/A, A/C, A/U and C/A, C/C, and C/U pairings isosteric with sheared A/G, as well as A/A and A/C pairings isosteric with both G/U and G/G bifurcated pairings. Thus, each non-Watson-Crick pair could be characterized by a phylogenetic signature of variations between isosteric-like pairings. In addition to the conservative changes, which form a dictionary of pairings isosterically compatible with those observed in the crystal structure, concerted changes involving several base pairs also occur. The latter covariations may indicate transitions between related but distinctive motifs within the loop E of 5S ribosomal RNA.     

Molecular dynamics study of intrinsic stability in six RNA terminal loop motifs

Single stranded RNA molecules can assume a wide range of tertiary structures beyond the canonical A-form double helix. Certain sequences, termed motifs, are more common than a random distribution would suggest. The existence of such motifs can be rationalized in structural terms. In this study, we have investigated the intrinsic structural stability of RNA terminal loop motifs using multiple MD simulations in explicit water. Representative loops were chosen from the major tetraloop motifs, including also the U-turn motif. Not all loops retain their folded starting structure, but lowering the temperature to 277 K, or adding adjacent base pairs from the stem to which the motif is attached, helps stabilizing the folded loop structure.     

Conformational dynamics of a 5S rRNA hairpin domain containing loop D and a single nucleotide bulge

Molecular modeling and molecular dynamics have been employed to study the conformation and flexibility of a 15-nucleotide fragment of the plant 5S rRNA containing loop D and a single uridine bulge. Two different model built initial structures were used: one with the bulge localized inside the helical stem and another with the bulge pointing out from the helix. Several independent 700-ps-long trajectories in aqueous solution with Na(+) conterions were produced for each starting structure. The bulge nucleotide inside the helix stayed in two main conformations, both of which affected the geometry of the stem part opposite the bulge. When the bulge nucleotide was located outside the helix, we found high base mobility and local backbone flexibility. The dynamics of the hydrogen bond network and conformational changes from a direct to a water mediated hydrogen bond in the sheared G-A basepair in the tetraloop was described. Our results correlate with lead ion induced cleavage patterns in 5S rRNA. Sites resistant to nonspecific lead cleavage appeared in all our simulations as the most rigid fragments independent of the localization of the bulge nucleotide.     

The solution structure of the loop E region of the 5S rRNA from spinach chloroplasts

A structure has been obtained for the loop E region of the 5S rRNA from Spinacia oleracia chloroplast ribosomes using residual dipolar coupling data as well as NOE, J coupling and chemical shift information. Even though the loop E sequence of this chloroplast 5S rRNA differs from that of Escherichia coli loop E at approximately 40% of its positions, its conformation is remarkably similar to that of E.coli loop E. Consistent with this conclusion, ribosomal protein L25 from E.coli, which binds to the loop E region of both intact E.coli 5S rRNA and to oligonucleotides containing that sequence, also binds to the chloroplast-derived oligonucleotide discussed here.     

Cations and hydration in catalytic RNA: molecular dynamics of the hepatitis delta virus ribozyme

The hepatitis delta virus (HDV) ribozyme is an RNA enzyme from the human pathogenic HDV. Cations play a crucial role in self-cleavage of the HDV ribozyme, by promoting both folding and chemistry. Experimental studies have revealed limited but intriguing details on the location and structural and catalytic functions of metal ions. Here, we analyze a total of approximately 200 ns of explicit-solvent molecular dynamics simulations to provide a complementary atomistic view of the binding of monovalent and divalent cations as well as water molecules to reaction precursor and product forms of the HDV ribozyme. Our simulations find that an Mg2+ cation binds stably, by both inner- and outer-sphere contacts, to the electronegative catalytic pocket of the reaction precursor, in a position to potentially support chemistry. In contrast, protonation of the catalytically involved C75 in the precursor or artificial placement of this Mg2+ into the product structure result in its swift expulsion from the active site. These findings are consistent with a concerted reaction mechanism in which C75 and hydrated Mg2+ act as general base and acid, respectively. Monovalent cations bind to the active site and elsewhere assisted by structurally bridging long-residency water molecules, but are generally delocalized.     

Automated identification of RNA conformational motifs: theory and application to the HM LSU 23S rRNA

We develop novel methods for recognizing and cataloging conformational states of RNA, and for discovering statistical rules governing those states. We focus on the conformation of the large ribosomal subunit from Haloarcula marismortui. The two approaches described here involve torsion matching and binning. Torsion matching is a pattern-recognition code which finds structural repetitions. Binning is a classification technique based on distributional models of the data. In comparing the results of the two methods we have tested the hypothesis that the conformation of a very large complex RNA molecule can be described accurately by a limited number of discrete conformational states. We identify and eliminate extraneous and redundant information without losing accuracy. We conclude, as expected, that four of the torsion angles contain the overwhelming bulk of the structural information. That information is not significantly compromised by binning the continuous torsional information into a limited number of discrete values. The correspondence between torsion matching and binning is 99% (per residue). Binning, however, does have several advantages. In particular, we demonstrate that the conformation of a large complex RNA molecule can be represented by a small alphabet. In addition, the binning method lends itself to a natural graphical representation using trees.     

RNA kink-turns as molecular elbows: hydration, cation binding, and large-scale dynamics

The presence of Kink-turns (Kt) at key functional sites in the ribosome (e.g., A-site finger and L7/L12 stalk) suggests that some Kink-turns can confer flexibility on RNA protuberances that regulate the traversal of tRNAs during translocation. Explicit solvent molecular dynamics demonstrates that Kink-turns can act as flexible molecular elbows. Kink-turns are associated with a unique network of long-residency static and dynamical hydration sites that is intimately involved in modulating their conformational dynamics. An implicit solvent conformational search confirms the flexibility of Kink-turns around their X-ray geometries and identifies a second low-energy region with open structures that could correspond to Kink-turn geometries seen in solution experiments. An extended simulation of Kt-42 with the factor binding site (helices 43 and 44) shows that the local Kt-42 elbow-like motion fully propagates beyond the Kink-turn, and that there is no other comparably flexible site in this rRNA region. Kink-turns could mediate large-scale adjustments of distant RNA segments.     

Localization of 5S RNA and rRNA genes in the Norway rat

The genes coding for the two classes of ribosomal RNA molecules, 5S RNA and 18+28S RNA, have been localized in the Norway rat (Rattus norvegicus). The 18+28S RNA cistrons are found on three chromosomes, at secondary constrictions on the short arms of chromosomes 3 and 12 and at the telomere of the short arm of chromosome 11. These sites were confirmed using the silver staining technique for nucleolar organizer regions. Two sites were found for the 5S RNA genes; one is closely linked to the 18+28S gene site on chromosome 12. The second site is at or near the telomere of the long arm of chromosome 19.     

Elastic properties of ribosomal RNA building blocks: molecular dynamics of the GTPase-associated center rRNA

Explicit solvent molecular dynamics (MD) was used to describe the intrinsic flexibility of the helix 42–44 portion of the 23S rRNA (abbreviated as Kt-42+rGAC; kink-turn 42 and GTPase-associated center rRNA). The bottom part of this molecule consists of alternating rigid and flexible segments. The first flexible segment (Hinge1) is the highly anharmonic kink of Kt-42. The second one (Hinge2) is localized at the junction between helix 42 and helices 43/44. The rigid segments are the two arms of helix 42 flanking the kink. The whole molecule ends up with compact helices 43/44 (Head) which appear to be modestly compressed towards the subunit in the Haloarcula marismortui X-ray structure. Overall, the helix 42–44rRNA is constructed as a sophisticated intrinsically flexible anisotropic molecular limb. The leading flexibility modes include bending at the hinges and twisting. The Head shows visible internal conformational plasticity, stemming from an intricate set of base pairing patterns including dynamical triads and tetrads. In summary, we demonstrate how rRNA building blocks with contrasting intrinsic flexibilities can form larger architectures with highly specific patterns of preferred low-energy motions and geometries.     

Coevolution in RNA Molecules Driven by Selective Constraints: Evidence from 5S rRNA

Understanding intra-molecular coevolution helps to elucidate various structural and functional constraints acting on molecules and might have practical applications in predicting molecular structure and interactions. In this study, we used 5S rRNA as a template to investigate how selective constraints have shaped the RNA evolution. We have observed the nonrandom occurrence of paired differences along the phylogenetic trees, the high rate of compensatory evolution, and the high TIR scores (the ratio of the numbers of terminal to intermediate states), all of which indicate that significant positive selection has driven the evolution of 5S rRNA. We found three mechanisms of compensatory evolution: Watson-Crick interaction (the primary one), complex interactions between multiple sites within a stem, and interplay of stems and loops. Coevolutionary interactions between sites were observed to be highly dependent on the structural and functional environment in which they occurred. Coevolution occurred mostly in those sites closest to loops or bulges within structurally or functionally important helices, which may be under weaker selective constraints than other stem positions. Breaking these pairs would directly increase the size of the adjoining loop or bulge, causing a partial or total structural rearrangement. In conclusion, our results indicate that sequence coevolution is a direct result of maintaining optimal structural and functional integrity.     

The nucleotide sequence of ribosomal 5S RNA from Rhizobium meliloti: comparison with 5S rRNA of Agrobacterium

The complete nucleotide sequence of R. meliloti 5S ribosomal RNA has been determined and compared with the already known sequence of A. tumefaciens 5S rRNA (Vandenberghe et al., 1985, Eur. J. Biochem., 149, 537-542) and of other 5S rRNAs from Rodobacteria Alpha-2 (Wolters et al., 1988, Nucleic Acids Res., 16, rl-r70). The differences found at eight positions (23, 73, 83, 72 in helical fragments; 16, 40, 88 in loops; 54 in bulge), which might affect secondary structures of 5S rRNA, are small. Moreover, the sequence analysis specifies both variable and common positions in 5S rRNA secondary structure of Rodobacteria Alpha-2.     

Identification and role of functionally important motifs in the 970 loop of Escherichia coli 16S ribosomal RNA

The 970 loop (helix 31) of Escherichia coli 16S ribosomal RNA contains two modified nucleotides, m²G966 and m⁵C967. Positions A964, A969, and C970 are conserved among the Bacteria, Archaea, and Eukarya. The nucleotides present at positions 965, 966, 967, 968, and 971, however, are only conserved and unique within each domain. All organisms contain a modified nucleoside at position 966, but the type of the modification is domain specific. Biochemical and structure studies have placed this loop near the P site and have shown it to be involved in the decoding process and in binding the antibiotic tetracycline. To identify the functional components of this ribosomal RNA hairpin, the eight nucleotides of the 970 loop of helix 31 were subjected to saturation mutagenesis and 107 unique functional mutants were isolated and analyzed. Nonrandom nucleotide distributions were observed at each mutated position among the functional isolates. Nucleotide identity at positions 966 and 969 significantly affects ribosome function. Ribosomes with single mutations of m²G966 or m⁵C967 produce more protein in vivo than do wild-type ribosomes. Overexpression of initiation factor 3 specifically restored wild-type levels of protein synthesis to the 966 and 967 mutants, suggesting that modification of these residues is important for initiation factor 3 binding and for the proper initiation of protein synthesis.     

Organization of the ribosomal RNA genes in Mycoplasma hyopneumoniae: the 5S rRNA gene is separated from the 16S and 23S rRNA genes

Summary In order to study the organization of the ribosomal RNA genes of Mycoplasma hyopneumoniae the rRNA genes were cloned in phage vectors EMBL3 and EMBL4. By subcloning the restriction fragments into various plasmids and analysing the resulting clones by Southern and Northern blot hybridization, a restriction map of the rRNA genes was generated and the organization of the rRNA genes was determined. The results show that the genes for the 16S and 23S rRNAs are closely spaced and occur only once in the genome, whereas the 5S rRNA gene is separated from the other two genes by more than 4 kb.     

An E. coli 5S rRNA Deletion Mutant Useful for the Study of 5S rRNA Structure/Function Relationships

We report on the construction of a novel strain of E. coli that can be useful for studies on the structure/function relationship of 5S rRNAs. The bacterial strain is deficient in six of the eight naturally occurring 5S rRNA genes (operons B, D, H, G, E) and demonstrates a greatly reduced growth rate that can be compensated by the plasmid-encoded expression of 5S rRNA. The relatively large difference in growth rate between compensated and non-compensated mutants provides the basis for a quick and simple assaying system for both the evaluation and mass screening of divergent 5S rRNA sequences for function. We describe the construction of the 5S rRNA deletion mutant BDHGE and characterize the usefulness and limitations of the system for evaluating structure/function relationships of 5S rRNA sequence. Received: 20 August 2000 / Accepted: 2 January 2001     

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.