Biochemical and NMR spectroscopy evidence for a new tertiary A-U base pair in lupin ribosomal 5 S RNA structure.

The new model for the tertiary structure of ribosomal 5 S rRNA from plants recently proposed by some of us has been already supported by RNase H digestions in the presence of complementary oligodeoxynucleotides. These results are confirmed now by the new biochemical and NMR spectroscopy data. Diethylpyrocarbonate (DEP) and monoperphthalic acid (MPA) are the reagents with the high specificity toward single-stranded adenosine residues. Our experiments clearly show that under native conditions adenosine 100 (A100) of lupin 5 S rRNA is not available for reaction toward these reagents. However under denaturing conditions this residue reacts with DEP and MPA. The detailed analysis of the lupin 5 S rRNA by NMR spectra provide the data on the specific interaction of A100-U53. Thus, we have seen for the first time the NMR signal due to the A100-U53 tertiary base pair, which as we believe, stabilizes interactions between loops B and E.     

Three-dimensional model of Escherichia coli ribosomal 5 S RNA as deduced from structure probing in solution and computer modeling.

The conformation of Escherichia coli 5 S rRNA was investigated using chemical and enzymatic probes. The four bases were monitored at one of their Watson-Crick positions with dimethylsulfate (at C(N-3) and A(N-1], with a carbodiimide derivative (at G(N-1) and U(N-3] and with kethoxal (at G(N-1, N-2]. Position N-7 of purine was probed with diethylpyrocarbonate (at A(N-7] and dimethylsulfate (at G(N-7]. Double-stranded or stacked regions were tested with RNase V1 and unpaired guanine residues with RNase T1. We also used lead(II) that has a preferential affinity for interhelical and loop regions and a high sensitivity for flexible regions. Particular care was taken to use uniform conditions of salt, magnesium, pH and temperature for the different enzymatic chemical probes. Derived from these experimental data, a three dimensional model of the 5 S rRNA was built using computer modeling which integrates stereochemical constraints and phylogenetic data. The three domains of 5 S rRNA secondary structure fold into a Y-shaped structure that does not accommodate long-range tertiary interactions between domains. The three domains have distinct structural and dynamic features as revealed by the chemical reactivity and the lead(II)-induced hydrolysis: domain 2 (loop B/helix III/loop C) displays a rather weak structure and possesses dynamic properties while domain 3 (helix V/region E/helix IV/loop D) adopts a highly structured and overall helical conformation. Conserved nucleotides are not crucial for the tertiary folding but maintain an intrinsic structure in the loop regions, especially via non-canonical pairing (A.G, G.U, G.G, A.C, C.C), which can close the loops in a highly specific fashion. In particular, nucleotides in the large external loop C fold into an organized conformation leading to the formation of a five-membered loop motif. Finally, nucleotides at the hinge region of the Y-shape are involved in a precise array of hydrogen bonds based on a triple interaction between U14, G69 and G107 stabilizing the quasi-colinearity of helices II and V. The proposed tertiary model is consistent with the localization of the ribosomal protein binding sites and possesses strong analogy with the model proposed for Xenopus laevis 5 S rRNA, indicating that the Y-shape model can be generalized to all 5 S rRNAs.     

A comparison of the solution structures and conformational properties of the somatic and oocyte 5S rRNAs of Xenopus laevis

The secondary and tertiary structures of Xenopus oocyte and somatic 5S rRNAs were investigated using chemical and enzymatic probes. The accessibility of both RNAs towards single-strand specific nucleases (T1, T2, A and S1) and a helix-specific ribonuclease from cobra venom (RNase V1) was determined. The reactivity of nucleobase N7, N3 and N1 positions towards chemical probes was investigated under native (5 mM MgCl2, 100 mM KCl, 20 degrees C) and semi-denaturing (1 mM EDTA, 20 degrees C) conditions. Ethylnitrosourea was used to identify phosphates not reactive towards alkylation under native conditions. The results obtained confirm the presence of the five helical stems predicted by the consensus secondary structure model of 5S rRNA. The chemical reactivity data indicate that loops C and D are involved in a number of tertiary interactions, and loop E folds into an unusual secondary structure. A comparison of the data obtained for the two types of Xenopus 5S rRNA indicates that the conformations of the oocyte and somatic 5S rRNAs are very similar. However, the data obtained with nucleases under native conditions, and chemical probes under semi-denaturing conditions, reveal that helices III and IV in the somatic 5S rRNA are less stable than the same structures in oocyte 5S rRNA. Using chimeric 5S rRNAs, it was possible to demonstrate that the relative resistance of oocyte 5S rRNA to partial denaturation in 4 M urea is conferred by the five oocyte-specific nucleotide substitutions in loop B/helix III. In contrast, the superior stability of oocyte 5S rRNA in the presence of EDTA is related to a single C substitution at position 79.     

Tertiary structure and computer modeling of plant 5S ribosomal RNA

A new model of secondary and tertiary structure of higher plant 5S rRNA is proposed. It consists of three domains. Domain alpha includes stem I and loop A; domain beta contains stems II and III and loops B and C; domain gamma consists of stems IV and V and loops D and E. We propose that the domains beta and gamma adopt RNA-A like structure due to irregularities caused by the different in size internal loops B and E and the bulges occurring in the model. A suggested bending of RNA could bring single stranded fragments of domains beta and gamma close enough to each other to allow tertiary interactions. The new model of plant 5S rRNA differs from those suggested previously for eukaryotic 5S rRNA, by arrangement of the domains beta and gamma and the base pairing scheme of domain gamma. The model is based on our results of partial digestion obtained with single and double strand specific nucleases. The experimental results were confirmed by computer aided secondary structure prediction analysis of all higher plant 5S rRNAs and computer modeling using energy minimalization approach. Further support of our model have been provided by experiments including alpha sarcin, ribonuclease H and chemical modifications.     

Comparative calorimetric studies on the dynamic conformation of plant 5S rRNA: II. Structural interpretation of the thermal unfolding patterns for lupin seeds and wheat germ.

Thermal unfolding of 5S rRNA from wheat germ (WG) and lupin seeds (LS) was studied in solution. Experimental curves of differential scanning calorimetry (DSC) were resolved into particular components according to the thermodynamic model of two-state transitions. The DSC temperature profiles for WG and LS differ significantly in spite of very high similarities in the sequence of both molecules. Those results are interpreted according to a model of the secondary and tertiary molecular structure of 5S rRNA. A comparison of the 'nearest neighbour' model of interaction with the experimental thermodynamic results enables a complete interpretation of the process of the melting of its structures. In light of our observations, the crucial differences between both DSC melting profiles are mainly an outcome of different thermodynamic properties of the first helical fragment 'A' made up of 9 complementary base pairs. It contains 6 differences in the nucleotide sequence of both types of molecules, which still retain 9-meric double helixes. The temperature stability of his helix in WG is much lower than of the LS one. Moreover, the results supply evidence for a strong specific tertiary interaction between the two hairpin loops 'c' and 'e' in both 5S rRNA molecules, modulated by small differences in the thermodynamic properties of both 5S rRNA.     

Computer modeling from solution data of spinach chloroplast and of Xenopus laevis somatic and oocyte 5 S rRNAs

Detailed atomic models of a eubacterial 5 S rRNA (spinach chloroplast 5 S rRNA) and of a eukaryotic 5 S rRNA (somatic and oocyte 5 S rRNA from Xenopus laevis) were built using computer graphic. Both models integrate stereochemical constraints and experimental data on the accessibility of bases and phosphates towards several structure-specific probes. The base sequence was first inserted on to three-dimensional structural fragments picked up in a specially devised databank. The fragments were modified and assembled interactively on an Evans & Sutherland PS330. Modeling was finalized by stereochemical and energy refinement. In spite of some uncertainty in the relative spatial orientation of the substructures, the broad features of the models can be generalized and several conclusions can be reached: (1) both models adopt a distorted Y-shape structure, with helices B and D not far from colinearity; (2) no tertiary interactions exist between loop c and region d or loop e; (3) the internal loops, in particular region d, contain several non-canonical base-pairs of A.A, U.U and A.G types; (4) invariant residues appear to be more important for protein or RNA binding than for maintaining the tertiary structure. The models are corroborated by footprinting experiments with ribosomal proteins and by the analysis of various mutants. Such models help to clarify the structure-function relationship of 5 S rRNA and are useful for designing site-directed mutagenesis experiments.     

New model of tertiary structure of plant 5S rRNA is confirmed by digestions with alpha-sarcin

The cytotoxin alpha-sarcin was employed to test the model of secondary and tertiary structures of plant 5S rRNAs, which we recently proposed [(1990) Int. J. Biol. Macromol. (in press)]. alpha-Sarcin is a novel ribonuclease that hydrolyzes phosphodiester bonds adjacent to purines in nucleic acids. The digestion pattern obtained for lupin and wheat germ 5S rRNAs strongly suggests the existence of tertiary interactions between residues C34, C35, C36, A37 and G85, G86, G87, U88 as previously proposed. The results on the secondary structure of plant 5S rRNA are in line with a previously proposed model.     

Higher plant 5S rRNAs share common secondary and tertiary structure. A new three domains model

A new model of secondary and tertiary structure of higher plant 5S RNA is proposed. It consists of three helical domains: domain alpha includes stem I; domain beta contains stems II and III and loops B and C; domain gamma consists of stems IV and V and loops D and E. Except for, presumably, a canonical RNA-A like domain alpha, the two remaining domains apparently adopt a perturbed RNA-A structure due to irregularities within internal loops B and E and three bulges occurring in the model. Bending of RNA could bring loops B and E and/or C and D closer making tertiary interactions likely. The model differs from that suggested for eukaryotic 5S rRNA, by organization of domain gamma. Our model is based on the results of partial digestion obtained with single- and double-strand RNA specific nucleases. The proposed secondary structure is strongly supported by the observation that crude plant 5S rRNA contains abundant RNA, identified as domain gamma of 5S rRNA. Presumably it is excised from the 5S rRNA molecule by a specific nuclease present in lupin seeds. Experimental results were confirmed by computer-aided secondary structure prediction analysis of all higher plant 5S rRNAs. Differences observed between earlier proposed models and our proposition are discussed.     

Application of a nuclease from rye nucleus for structural studies of plant ribonucleic acids.

A new nuclease (Rn) isolated from rye nucleus was applied for the structural studies of methionine initiator transfer ribonucleic acid and ribosomal 5S rRNA from yellow lupin seeds. The enzyme shows high specificity for some regions of both RNAs. The dihydrouridine and ribothymidine loops which are supposed to be involved in the tertiary interactions of the methionine initiator tRNA were hydrolysed. The anticodon loop is not digested at all. 5S rRNA was digested in single stranded regions (loops). The cleavage pattern of the tRNA and 5S rRNA obtained with Rn enzyme, suggests not only the high specificity toward single stranded regions, but also some dependence on their tertiary structure.     

Structural similarity of E. coli 5S rRNA in solution and within the ribosome

The article presents translational and rotational diffusion coefficients of 5S rRNA determined experimentally by the method of dynamic light scattering (DLS) and its comparison with the values predicted for different models of this molecule. The tertiary structure of free 5S rRNA was proposed on the basis of the atomic structures of the 5S rRNA from E. coli and H. marismortui extracted from the ribosome. A comparison of the values of DT, tauR, and Rg predicted for different models with experimental results for the free molecule in solution suggests that free 5S rRNA is less compact than that in the complex with ribosomal proteins. In general, the molecules of 5S rRNA consist of three domains: a short one and two longer ones. As follows from a comparison of the results of our simulations with experimental values, in the molecule in solution the two closest helical fragments of the longer domains remain collinear, whereas the short domain takes a position significantly deviated from them.     

The nucleotide sequence of 5S rRNA from bovine liver.

We have determined the nucleotide sequence of ribosomal 5S RNA from bovine liver. The comparison of this sequence with those from other eukaryotic sources shows that a common secondary structure model for all eukaryotic 5S rRNAs may exist. Analysis of the evolutionary conserved nucleotides in metazoan 5S rRNAs suggests that the tertiary interactions, proposed earlier for plant 5S rRNA, are also possible.     

Structural analysis of prokaryotic and eukaryotic 5S rRNAs by RNase H

The availabilities of single-stranded 5S rRNA regions c, d and d' for base pairing interactions were analyzed by using synthetic DNA oligomers. Hybrid formation was detected by the endonucleolytical mode of the RNA-DNA specific action of RNase H. Provided that the hybrid interaction involved 6 successive base pairs, 5S rRNA loop c nucleotides 42-47 displayed accessibility in Escherichia coli, Bacillus stearothermophilus and Thermus thermophilus 5S rRNAs as well as in eukaryotic 5S rRNAs from Saccharomyces carlsbergensis, Rattus rattus and Equisetum arvense. Investigating eubacterial 5S rRNA regions d and d' (nucleotides 71-76 and 99-105, respectively), susceptibility was observed in E. coli 5S rRNA which, however, decreases in B. stearothermophilus and even more so in T. thermophilus 5S rRNA. For additional evaluation of the data obtained by RNase H cleavage, association constants of the hexanucleotides were determined by equilibrium dialysis at 4 degrees C for B. stearothermophilus 5S rRNA. The results obtained reveal that nucleotides 36-41 of B. stearothermophilus 5S rRNA are inaccessible for Watson-Crick interaction, which suggests that this part of loop c is in a structurally constrained configuration, or buried in the tertiary structure or involved in tertiary interactions.     

Improved procedure for the isolation of a double-strand-specific ribonuclease and its application to structural analysis of various 5S rRNAs and tRNAs

An improved method for the isolation of a double-strand-specific RNase from snake venom is presented. This RNase, called CSV, was used to cleave yeast tRNAPhe and tRNA2Glu and tRNAfMet from Escherichia coli. In addition these RNAs and E. coli tRNAPhe were examined with the single-strand-specific nuclease S1. The results are discussed in terms of the specificity of CSV RNase and the structure of tRNAs. S1 nuclease digestions at increasing temperatures allowed the melting of tertiary and secondary structure to be monitored. 5S rRNA from E. coli, Thermoplasma acidophilum and the chloroplasts of Spinacia oleracea were digested with CSV and S1. The information these results give on the secondary-structural differences between different classes of 5S rRNA are discussed. Supporting evidence is found for tertiary interactions between hairpin loop c and internal loop d of eubacterial 5S rRNA.     

Studies on the structure and function of 16S ribosomal RNA using structure-specific chemical probes

Recent technological developments permit us to examine the accessibility of specific atoms on any nucleotide in any large RNA molecule to certain chemical probes. This can provide detailed information about the higher order structure of large RNA molecules, including secondary and tertiary structure, protein-RNA contacts, binding sites for functional ligands and possible biologically significant conformational changes. Here, we summarize recent studies on (i) the conformation of naked 16S rRNA under a variety of ionic conditions, and (ii) the behaviour of 16S rRNA in active and inactive 30S subunits, as defined by Zamir, Elson and their colleagues. The latter study reveals a reciprocal conformational change in the vicinity of the decoding region of 16S rRNA in 30S ribosomal subunits. This conformational change appears to be a rearrangement of tertiary and/or quaternary structure involving several universally conserved nucleotides. No reproducible effects are seen elsewhere in the molecule, suggesting that the active-inactive transition is a result of the observed conformational change.     

Mapping the three-dimensional locations of ribosomal RNA and proteins.

Seven regions of 16S rRNA have been located on the surface of the 30S ribosomal subunit by DNA hybridization electron microscopy in our laboratory. In addition, we have recently mapped the three-dimensional locations of an additional seven small ribosomal proteins by immunoelectron microscopy. The information from the direct mapping of the sites on rRNA has been incorporated into a model for the tertiary structure of 16S rRNA, accounting for approximately 40% of the total 16S rRNA. A novel structure, the platform ring, is proposed for a region of rRNA within the central domain. This structure rings the edges of the platform and includes regions 655-751 and 769-810. Another region, the recognition complex, consists of nucleotides 500-545, and occupies a region on the exterior surface of the subunit, near the EF-Tu binding site. In addition, 19 of the 21 small subunit ribosomal proteins have been mapped by immunoelectron microscopy in our laboratory. In order to evaluate the reliability of our model for the three-dimensional distribution of 16S rRNA, we have predicted which sites of rRNA are adjacent to ribosomal proteins and compared these predictions with r-protein protection studies of others. Good correlation between the model, the locations of rRNA sites, the locations of ribosomal proteins, and regions of rRNA protected by ribosomal proteins, provides independent support for this model.     

SARNA-Predict: accuracy improvement of RNA secondary structure prediction using permutation-based simulated annealing

Ribonucleic acid (RNA), a single-stranded linear molecule, is essential to all biological systems. Different regions of the same RNA strand will fold together via base pair interactions to make intricate secondary and tertiary structures that guide crucial homeostatic processes in living organisms. Since the structure of RNA molecules is the key to their function, algorithms for the prediction of RNA structure are of great value. In this article, we demonstrate the usefulness of SARNA-Predict, an RNA secondary structure prediction algorithm based on Simulated Annealing (SA). A performance evaluation of SARNA-Predict in terms of prediction accuracy is made via comparison with eight state-of-the-art RNA prediction algorithms: mfold, Pseudoknot (pknotsRE), NUPACK, pknotsRG-mfe, Sfold, HotKnots, ILM, and STAR. These algorithms are from three different classes: heuristic, dynamic programming, and statistical sampling techniques. An evaluation for the performance of SARNA-Predict in terms of prediction accuracy was verified with native structures. Experiments on 33 individual known structures from eleven RNA classes (tRNA, viral RNA, antigenomic HDV, telomerase RNA, tmRNA, rRNA, RNaseP, 5S rRNA, Group I intron 23S rRNA, Group I intron 16S rRNA, and 16S rRNA) were performed. The results presented in this paper demonstrate that SARNA-Predict can out-perform other state-of-the-art algorithms in terms of prediction accuracy. Furthermore, there is substantial improvement of prediction accuracy by incorporating a more sophisticated thermodynamic model (efn2).     

The spacing between functional Cis-elements of U3 snoRNA is critical for rRNA processing

The sequences and structural features of Xenopus laevis U3 small nucleolar RNA (snoRNA) necessary for pre-rRNA cleavage at sites 1 and 2 to form 18 S rRNA were assayed by depletion/rescue experiments in Xenopus oocytes. Mutagenesis results demonstrated that the putative stem of U3 domain I is unnecessary for 18 S rRNA processing. A model consistent with earlier experimental data is proposed for the structure of domain I when U3 is not yet bound to pre-rRNA. For its function in rRNA processing, a newly discovered element (5' hinge) was revealed to be important but not as critical as the 3' hinge region in Xenopus U3 snoRNA for 18 S rRNA formation. Base-pairing is proposed to occur between the U3 5' hinge and 3' hinge and complementary regions in the external transcribed spacer (ETS); these interactions are phylogenetically conserved, and are homologous to those previously described in yeast (5' hinge-ETS) and trypanosomes (3' hinge-ETS). A model is presented where the base-pairing of the 5' hinge and 3' hinge of U3 snoRNA with the ETS of pre-rRNA helps to correctly position U3 boxes A'+A for their function in rRNA processing. Like an earlier proposal for yeast, boxes A' and A of Xenopus may base-pair with 18 S sequences in pre-rRNA. We present the first direct experimental evidence in any system that box A' is essential for U3 snoRNA function in 18 S rRNA formation. The analysis of insertions and deletions indicated that the spacing between the U3 elements is important, suggesting that they base-pair with the ETS and 18 S regions of pre-rRNA at the same time.