An overview of the secondary structure of the V4 region of eukaryotic small-subunit ribosomal RNA.

The V4 region of the small subunit (18S) ribosomal RNA was examined in 72 different sequences representing a broad sample eukaryotic diversity. This domain is the most variable region of the 18S rRNA molecule and ranges in length from ca. 230 to over 500 bases. Based upon comparative analysis, secondary structural models were constructed for all sequences and the resulting generalized model shows that most organisms possess seven helices for this region. The protists and two insects show from one to as many as four helices in addition to the above seven. In this report, we summarize secondary structure information presented elsewhere for the V4 region, describe the general features for helical and apical regions, and identify signature sequences useful in helix identification. Our model generally agrees with other current concepts; however, we propose modifications or alternative structures for the start of the V4 region, the large protist inserts, and the sector that may possibly contain a pseudoknot.     

The evolution of 5S RNA secondary structures

We have applied the Pipas-McMahon algorithm based on free energy calculations to the search for a 5S RNA base-pair structure common to all known sequences. We find that a 'Y' shaped model is consistently among the structures having the lowest free energy using 5S RNA sequences from either eukaryotic or prokaryotic sources. Compaison of this 'Y' structure with models which have recently been proposed show these models to be remarkably similar, and the minor differences are explicable based on the technique used to obtain the model. That prokaryotic and eukaryotic 5S RNA can adopt a similar secondary structure is strong support for its resistance to change during evolution.     

Prediction of RNA secondary structure based on helical regions distribution

MOTIVATION: RNAs play an important role in many biological processes and knowing their structure is important in understanding their function. Due to difficulties in the experimental determination of RNA secondary structure, the methods of theoretical prediction for known sequences are often used. Although many different algorithms for such predictions have been developed, this problem has not yet been solved. It is thus necessary to develop new methods for predicting RNA secondary structure. The most-used at present is Zuker's algorithm which can be used to determine the minimum free energy secondary structure. However many RNA secondary structures verified by experiments are not consistent with the minimum free energy secondary structures. In order to solve this problem, a method used to search a group of secondary structures whose free energy is close to the global minimum free energy was developed by Zuker in 1989. When considering a group of secondary structures, if there is no experimental data, we cannot tell which one is better than the others. This case also occurs in combinatorial and heuristic methods. These two kinds of methods have several weaknesses. Here we show how the central limit theorem can be used to solve these problems. RESULTS: An algorithm for predicting RNA secondary structure based on helical regions distribution is presented, which can be used to find the most probable secondary structure for a given RNA sequence. It consists of three steps. First, list all possible helical regions. Second, according to central limit theorem, estimate the occurrence probability of every helical region based on the Monte Carlo simulation. Third, add the helical region with the biggest probability to the current structure and eliminate the helical regions incompatible with the current structure. The above processes can be repeated until no more helical regions can be added. Take the current structure as the final RNA secondary structure. In order to demonstrate the confidence of the program, a test on three RNA sequences: tRNAPhe, Pre-tRNATyr, and Tetrahymena ribosomal RNA intervening sequence, is performed. AVAILABILITY: The program is written in Turbo Pascal 7.0. The source code is available upon request. CONTACT: Wujj@nic.bmi.ac.cn or Liwj@mail.bmi.ac.cn      

A comparison of the crystal structures of eukaryotic and bacterial SSU ribosomal RNAs reveals common structural features in the hypervariable regions

While the majority of the ribosomal RNA structure is conserved in the three major domains of life--archaea, bacteria, and eukaryotes, specific regions of the rRNA structure are unique to at least one of these three primary forms of life. In particular, the comparative secondary structure for the eukaryotic SSU rRNA contains several regions that are different from the analogous regions in the bacteria. Our detailed analysis of two recently determined eukaryotic 40S ribosomal crystal structures, Tetrahymena thermophila and Saccharomyces cerevisiae, and the comparison of these results with the bacterial Thermus thermophilus 30S ribosomal crystal structure: (1) revealed that the vast majority of the comparative structure model for the eukaryotic SSU rRNA is substantiated, including the secondary structure that is similar to both bacteria and archaea as well as specific for the eukaryotes, (2) resolved the secondary structure for regions of the eukaryotic SSU rRNA that were not determined with comparative methods, (3) identified eukaryotic helices that are equivalent to the bacterial helices in several of the hypervariable regions, (4) revealed that, while the coaxially stacked compound helix in the 540 region in the central domain maintains the constant length of 10 base pairs, its two constituent helices contain 5+5 bp rather than the 6+4 bp predicted with comparative analysis of archaeal and eukaryotic SSU rRNAs.     

A universal model for the secondary structure of 5.8S ribosomal RNA molecules, their contact sites with 28S ribosomal RNAs, and their prokaryotic equivalent

The phylogenetic approach (ref. 1) has been utilized in construction of a universal 5.8S rRNA secondary structure model, in which about 65% of the residues exist in paired structures. Conserved nucleotides primarily occupy unpaired regions. Multiple compensating base changes are demonstrated to be present in each of the five postulated helices, thereby forming a major basis for their proof. The results of chemical and enzymatic probing of 5.8S rRNAs (ref. 13, 32) are fully consistent with, and support, our model. This model differs in several ways from recently proposed 5.8S rRNA models (ref. 3, 4), which are discussed. Each of the helices in our model has been extended to the corresponding bacterial, chloroplast and mitochondrial sequences, which are demonstrated to be positionally conserved by alignment with their eukaryotic counterparts. This extension is also made for the base paired 5.8S/28S contact points, and their prokaryotic and organelle counterparts. The demonstrated identity of secondary structure in these diverse molecules strongly suggests that they perform equivalent functions in prokaryotic and eukaryotic ribosomes.     

Refined secondary structure models for the 16S and 23S ribosomal RNA of Escherichia coli

The complete range of published sequences for ribosomal RNA (or rDNA), totalling well over 50,000 bases, has been used to derive refined models for the secondary structures of both 16S and 23S RNA from E. coli. Particular attention has been paid to resolving the differences between the various published secondary structures for these molecules. The structures are described in terms of 133 helical regions (45 for 16S RNA and 88 for 23S RNA). Of these, approximately 20 are still tentative or unconfirmed. A further 20 represent helical regions which definitely exist, but where the detailed base-pairing is still open to discussion. Over 90 of the helical regions are however now precisely established, at least to within one or two base pairs.     

The sequence of the 5.8 S ribosomal RNA of the crustacean Artemia salina. With a proposal for a general secondary structure model for 5.8 S ribosomal RNA.

We report the primary structure of 5.8 S rRNA from the crustacean Artemia salina. The preparation shows length heterogeneity at the 5'-terminus, but consists of uninterrupted RNA chains, in contrast to some insect 5.8 S rRNAs, which consist of two chains of unequal length separated in the gene by a short spacer. The sequence was aligned with those of 11 other 5.8 S rRNAs and a general secondary structure model derived. It has four helical regions in common with the model of Nazar et al. (J. Biol. Chem. 250, 8591-8597 (1975)), but for a fifth helix a different base pairing scheme was found preferable, and the terminal sequences are presumed to bind to 28 S rRNA instead of binding to each other. In the case of yeast, where both the 5.8 S and 26 S rRNA sequences are known, the existence of five helices in 5.8 S rRNA is shown to be compatible with a 5.8 S - 26 S rRNA interaction model.     

Putative secondary structures of unusually long strepsipteran SSU rRNAs and its phylogenetic implications.

We constructed the putative secondary structures of the small subunit rRNAs (SSU rRNA) from three strepsipteran insects. The primary sequences of the strepsipteran SSU rRNAs are unusually long due to unique and long insertions. In spite of these insertions, the basic shapes of their secondary structures are well maintained as shown in those of other eukaryotes, because these insertions appear mainly in the variable regions. The secondary structures for the V1, V3, V5, V8, and V9 regions are well conserved, even though the primary structures of V1, V5, and V8 regions are quite variable. However, the predicted secondary structures for the V2, V4, and V7 regions are quite different from those of other insects. In the V4 and V7 regions, helices specific to the Strepsiptera exist. These helices have not been reported in other organisms so far. Similarly, four eukaryotic specific helices (E8-1, E10-2, E23-4 and E45-1) not reported in insects exist in the V2, V4, and V8 regions. These helices are formed by the inserted sequences. The secondary structures of the expanded segments of the strepsipteran SSU rRNA were applied to infer the phylogenetic position of Strepsiptera, one of the most enigmatic problems in insect phylogeny. Only the secondary structure of the V7 region showed the weak Strepsiptera/Diptera sister-group relationship.     

Secondary structure models of the nuclear internal transcribed spacer regions and 5.8S rRNA in Calciodinelloideae (Peridiniaceae) and other dinoflagellates

Secondary structure models of the 5.8S rRNA and both internal transcribed spacers (ITS1 and ITS2) are proposed for Calciodinelloideae (Peridiniaceae) and are also plausible for other dinoflagellates. The secondary structure of the 5.8S rRNA corresponds to previously developed models, with two internal paired regions and at least one 5.8S rRNA–28S rRNA interaction. A general secondary structure model of ITS1 for Calciodinelloideae (and other dinoflagellates), consisting of an open multibranch loop with three major helices, is proposed. The homology of these paired regions with those found in other taxa, published in previous studies (e.g. yeast, green algae and Platyhelmithes) remains to be determined. Finally, a general secondary structure model of ITS2 for Calciodinelloideae (and other dinoflagellates) is reconstructed. Based on the 5.8S rRNA–28S rRNA interaction, it consists of a closed multibranch loop, with four major helices. At least helix III and IV have homology with paired regions found in other eukaryotic taxa (e.g. yeast, green algae and vertebrates). Since the secondary structures of both ITS regions are more conserved than the nucleotide sequences, their analysis helps in understanding molecular evolution and increases the number of structural characters. Thus, the structure models developed in this study may be generally useful for future phylogenetic analyses.     

Sequences of three molluscan 5 S ribosomal RNAs confirm the validity of a dynamic secondary structure model.

The collection of known 5 S rRNA primary structures is enriched with the sequences from three mollusca, the snails Helix pomatia and Arion rufus, and the mussel Mytilus edulis. The three sequences can be fitted in a five-helix secondary structure model previously shown (De Wachter et al. (1982) Biochimie 64, 311-329) to apply to all 5 S RNAs regardless of their origin. One of the helices in this model can undergo a bulge-internal loop transition. Within the metazoan kingdom, the dimensions of each helix and loop are rigidly conserved, except for one helix which can comprise either 6 or 7 base pairs.     

Phylogenetic analysis of the family Thermaceae with an emphasis on signature position and secondary structure of 16S rRNA

The sequences of the 16S rRNA genes from 38 strains of the family Thermaceae were compared by alignment analysis. The genus-specific and species-specific base substitutions or base deletions (signature positions) were found in three hypervariable regions (in the helices 6, 10 and 17). The differentiation of secondary structures of the high variable regions in the 5' end (38-497) containing several signature positions further supported the concept. Based on the comparisons of the secondary structures in the segments of 16S rRNAs, a key to the species of the family Thermaceae was proposed.     

The stereochemistry of a four-way DNA junction: a theoretical study.

The stereochemical conformation of the four-way helical junction in DNA (the Holliday junction; the postulated central intermediate of genetic recombination) has been analysed, using molecular mechanical computer modelling. A version of the AMBER program package was employed, that had been modified to include the influence of counterions and a global optimisation procedure. Starting from an extended planar structure, the conformation was varied in order to minimise the energy, and we discuss three structures obtained by this procedure. One structure is closely related to a square-planar cross, in which there is no stacking interaction between the four double helical stems. This structure is probably closely similar to that observed experimentally in the absence of cations. The remaining two structures are based on related, yet distinct, conformations, in which there is pairwise coaxial stacking of neighbouring stems. In these structures, the four DNA stems adopt the form of two quasi-continuous helices, in which base stacking is very similar to that found in standard B-DNA geometry. The two stacked helices so formed are not aligned parallel to each other, but subtend an angle of approximately 60 degrees. The strands that exchange between one stacked helix and the other are disposed about the smaller angle of the cross (i.e. 60 degrees rather than 120 degrees), generating an approximately antiparallel alignment of DNA sequences. This structure is precisely the stacked X-structure proposed on the basis of experimental data. The calculations indicate distortions from standard B-DNA conformation that are required to adopt the stacked X-structure; a widening of the minor groove at the junction, and reorientation of the central phosphate groups of the exchanging strands. An important feature of the stacked X-structure is that it presents two structurally distinct sides. These may be recognised differently by enzymes, providing a rationalisation for the points of cleavage by Holliday resolvases.     

A proposal for a specific double-helical structure in which the polynucleotide strands intercalate instead of forming base-pairs

Double helices, since the discovery of the DNA structure by Watson and Crick, represent the single most important secondary structural form of nucleic acids. The secondary structures of a variety of polynucleotide helices have now been well characterised with hydrogen-bonded base-pairs as building blocks. We wish to propose here the possibility, in a specific case, of a double stranded helical structure without any base-pair, but having a repeat unit of two nucleotides with their bases stacked through intercalation. The proposal comes from the initial models we have built for poly(dC) using the stacking patterns found in the crystal structures of 5'-dCMPNa2 which crystallises in two forms depending on the degree of hydration. These structures have pairs of nucleotides with the cytosine rings partially overlapping and separated by 3.3A. Using these as repeat units one could generate a model for poly(dC) with parallel strands, having a turn angle of 30 degrees and a base separation of 6.6A along each strand. Both right and left handed models with these parameters can be built in a smooth fashion without any obviously unreasonable stereochemical contacts. The helix diameter is about 13.5A, much smaller than that of normal helices with base-pair repeats. The changes in the sugar-phosphate backbone conformation in the present models compared to normal duplexes only reflect the torsional flexibility available for extension of polynucleotide chains as manifested by the crystal structures of drug-inserted oligonucleotide complexes. Intercalation proposed here could have some structural relevance elsewhere, for instance to the base-mismatched regions on the double helix and the packing of noncomplementary single strands as found in the filamentous bacteriophage Pf1.     

Nuclease S1 analysis of eubacterial 5S rRNA secondary structure

Summary Single-strand-specific nuclease S₁ was employed as a structural probe to confirm locations of unpaired nucleotide bases in 5S rRNAs purified from prokaryotic species of rRNA superfamily I. Limited nuclease S₁ digests of 3- and 5-end-labeled [³²P]5S rRNAs were electrophoresed in parallel with reference endoribonuclease digests on thin allel with reference endoribonuclease digests on thin sequencing gels. Nuclease S₁ primary hydrolysis patterns were comparable for 5S rRNAs prepared from all 11 species examined in this study. The locations of base-paired regions determined by enzymatic analysis corroborate the general features of the proposed universal five-helix model for prokaryotic 5S rRNA, although the results of this study suggest a significant difference between prokaryotic and eukaryotic 5S rRNAs in the evolution of helix IV. Furthermore, the extent of base-pairing predicted by helix IV needs to be reevaluated for eubacterial species. Clipping patterns in helices II and IV appear to be consistent with a secondary structural model that undergoes a conformational rearrangement between two (or more) structures. Primary clipping patterns in the helix II region, obtained by S₁ analysis, may provide useful information concerning the tertiary structure of the 5S rRNA molecule.     

Structures of the transmembrane helices of the G-protein coupled receptor, rhodopsin.

An hypothesis is tested that individual peptides corresponding to the transmembrane helices of the membrane protein, rhodopsin, would form helices in solution similar to those in the native protein. Peptides containing the sequences of helices 1, 4 and 5 of rhodopsin were synthesized. Two peptides, with overlapping sequences at their termini, were synthesized to cover each of the helices. The peptides from helix 1 and helix 4 were helical throughout most of their length. The N- and C-termini of all the peptides were disordered and proline caused opening of the helical structure in both helix 1 and helix 4. The peptides from helix 5 were helical in the middle segment of each peptide, with larger disordered regions in the N- and C-termini than for helices 1 and 4. These observations show that there is a strong helical propensity in the amino acid sequences corresponding to the transmembrane domain of this G-protein coupled receptor. In the case of the peptides from helix 4, it was possible to superimpose the structures of the overlapping sequences to produce a construct covering the whole of the sequence of helix 4 of rhodopsin. As similar superposition for the peptides from helix 1 also produced a construct, but somewhat less successfully because of the disordering in the region of sequence overlap. This latter problem was more severe for helix 5 and therefore a single peptide was synthesized for the entire sequence of this helix, and its structure determined. It proved to be helical throughout. Comparison of all these structures with the recent crystal structure of rhodopsin revealed that the peptide structures mimicked the structures seen in the whole protein. Thus similar studies of peptides may provide useful information on the secondary structure of other transmembrane proteins built around helical bundles.     

The secondary structure of human 28S rRNA: The structure and evolution of a mosaic rRNA gene

Summary We have determined the secondary structure of the human 28S rRNA molecule based on comparative analysis of available eukaryotic cytoplasmic and prokaryotic large-rRNA gene sequences. Examination of large-rRNA sequences of both distantly and closely related species has enabled us to derive a structure that accounts both for highly conserved sequence tracts and for previously unanalyzed variable-sequence tracts that account for the evolutionary differences in size among the large rRNAs.Human 28S rRNA is composed of two different types of sequence tracts: conserved and variable. They differ in composition, degree of conservation, and evolution. The conserved regions demonstrate a striking constancy of size and sequence. We have confirmed that the conserved regions of large-rRNA molecules are capable of forming structures that are superimposable on one another. The variable regions contain the sequences responsible for the 83% increase in size of the human large-rRNA molecule over that ofEscherichia coli. Their locations in the gene are maintained during evolution. They are G+C rich and largely nonhomologous, contain simple repetitive sequences, appear to evolve by frequent recombinational events, and are capable of forming large, stable hairpins.The secondary-structure model presented here is in close agreement with existing prokaryotic 23S rRNA secondary-structure models. The introduction of this model helps resolve differences between previously proposed prokaryotic and eukaryotic large-rRNA secondary-structure models.     

The tertiary structure of the four-way DNA junction affords protection against DNase I cleavage.

The accessibility of phosphodiester bonds in the DNA of four-way helical junctions has been probed with the nuclease DNase I. Regions of protection were observed on all four strands of the junctions, that tended to be longer on the strands that are exchanged between the coaxially stacked pairs of helices. The protected regions on the continuous strands of the stacked helices were not located exactly at the junction, but were displaced towards the 3' side of the strand. This is the region of backbone that becomes located in the major groove of the opposed helix in the non-crossed, right-handed structure for the junction, and might therefore be predicted to be protected against cleavage by an enzyme. However, the major grooves of the structure remain accessible to the much smaller probe dimethyl sulphate.     

The hydrodynamic shape, conformation, and molecular model of Escherichia coli ribosomal 5 S RNA.

The structure of ribosomal 5 S RNA has been examined using several physical biochemical techniques. Hydrodynamic measurements yield a s020,omega and [eta] of 5.5 x 10(-13) x and 6.9 ml/g, respectively. Other parameters calculated from these values indicate the shape of 5 S RNA is consistent with that of a prolate ellipsoid 160 A in length and 32 A wide. Sedimentation equilibrium results show that 5 S RNA exists as a monomer in the reconstitution buffer with an apparent molecular weight of 44,000. Ultraviolet absorption difference spectra show that approximately 75% of the bases in 5 S RNA are involved in base pairing, and of these base pairs 70% are G-C and 30% are A-U. These results on the overall shape and secondary structure of 5 S RNA have been incorporated with the results of other investigators as to the possible location of single-stranded and double-stranded helical regions, and a molecular model for 5 S RNA is proposed. The molecular model consists of three double helices in the shape of a prolate ellipsoid, with two of the double helical regions at one end of the molecule. The structure is consistent with the available data on the structure and function of 5 S RNA and bears similarity to the molecular model proposed by Osterberg et al. ((1976) Eur. J. Biochem. 68, 481-487) based on small angle x-ray scattering results and the secondary structure proposed by Madison ((1968) Annu. Rev. Biochem. 37, 131-148).     

A family of double helices of alternating poly(gamma-benzyl-D-L-glutamate), a stereochemical model for gramicidin A.

Poly(γ-benzyl-d-l-glutamate) with strict alternation of l and d residues is found to exist, in addition to the α_DL and π_DL^4.4 helical structures already described (Heitz et al., 1975a), in four more helical structures. Models based on double helices made of antiparallel strands are proposed for all four structures, based on infrared, X-ray and electron diffraction data. These double helices are, like the single-stranded π_DL helices, specific to polypeptides with a strict stereosequence of alternating l and d residues. The diameter of the helical core of three of these helices appears to depend on the dimensions of the solvent molecules. Conformational angles (located in the β regions) and atomic co-ordinates determined by conformational energy analysis are given for the four structures. Experimental conditions used to obtain these helices, and to induce transconformations between the various helical structures of PBd-lG are described. The present investigations on PBd-lG help to make more precise the structure and geometry of models proposed (Veatch et al., 1974) for the antibiotic gramicidin A.