The building blocks and motifs of RNA architecture

RNA motifs can be defined broadly as recurrent structural elements containing multiple intramolecular RNA-RNA interactions, as observed in atomic-resolution RNA structures. They constitute the modular building blocks of RNA architecture, which is organized hierarchically. Recent work has focused on analyzing RNA backbone conformations to identify, define and search for new instances of recurrent motifs in X-ray structures. One current view asserts that recurrent RNA strand segments with characteristic backbone configurations qualify as independent motifs. Other considerations indicate that, to characterize modular motifs, one must take into account the larger structural context of such strand segments. This follows the biologically relevant motivation, which is to identify RNA structural characteristics that are subject to sequence constraints and that thus relate RNA architectures to sequences.     

Refinement of the solution structure of a branched DNA three-way junction.

We have refined the structure of the DNA Three-Way Junction complex, TWJ-TC, described in the companion paper by quantitative analysis of two 2D NOESY spectra (mixing times 60 and 200 ms) obtained in D2O solution. NOESY crosspeak intensities extracted from these spectra were used in two kinds of refinement procedure: 1) distance-restrained energy minimization (EM) and molecular dynamics (MD) and 2) full relaxation matrix back calculation refinement. The global geometry of the refined model is very similar to that of a published, preliminary model (Leontis, 1993). Two of the helical arms of the junction are stacked. These are Helix 1, defined by basepairs S1-G1/S3-C12 through S1-C5/S3-G8 and Helix 2, which comprises basepairs S1-C6/S2-G5 through S1-G10/S2-G1. The third helical arm (Helix 3), comprised of basepairs S2-C6/S3-G5 through S2-C10/S3-G1 extends almost perpendicularly from the axis defined by Helices 1 and 2. The bases S1-C5 and S1-C6 of Strand 1 are continuously stacked across the junction region. The conformation of this strand is close to that of B-form DNA along its entire length, including the S1-C5 to S1-C6 dinucleotide step at the junction. The two unpaired bases S3-T6 and S3-C7 lie outside of the junction along the minor groove of Helix 1 and largely exposed to solvent. Analysis of the refined structure reveals that the glycosidic bond of S3-T6 exists in the syn conformation, allowing the methyl group of this residue to contact the hydrophobic surface of the minor groove of Helix 1, at S3-G11.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relative stabilities of DNA three-way, four-way and five-way junctions (multi-helix junction loops): unpaired nucleotides can be stabilizing or destabilizing.

Competition binding and UV melting studies of a DNA model system consisting of three, four or five mutually complementary oligonucleotides demonstrate that unpaired bases at the branch point stabilize three- and five-way junction loops but destabilize four-way junctions. The inclusion of unpaired nucleotides permits the assembly of five-way DNA junction complexes (5WJ) having as few as seven basepairs per arm from five mutually complementary oligonucleotides. Previous work showed that 5WJ, having eight basepairs per arm but lacking unpaired bases, could not be assembled [Wang, Y.L., Mueller, J.E., Kemper, B. and Seeman, N.C. (1991) Biochemistry, 30, 5667-5674]. Competition binding experiments demonstrate that four-way junctions (4WJ) are more stable than three-way junctions (3WJ), when no unpaired bases are included at the branch point, but less stable when unpaired bases are present at the junction. 5WJ complexes are in all cases less stable than 4WJ or 3WJ complexes. UV melting curves confirm the relative stabilities of these junctions. These results provide qualitative guidelines for improving the way in which multi-helix junction loops are handled in secondary structure prediction programs, especially for single-stranded nucleic acids having primary sequences that can form alternative structures comprising different types of junctions.     

cDNA cloning of a developmentally regulated hemocyanin subunit in the crustacean Cancer magister and phylogenetic analysis of the hemocyanin gene family

The complete cDNA sequence and protein reading frame of a developmentally regulated hemocyanin subunit in the Dungeness crab (Cancer magister) is presented. The protein sequence is aligned with 18 potentially homologous hemocyanin-type proteins displaying apparent sequence similarities. Functional domains are identified, and a comparison of predicted hydrophilicities, surface probabilities, and regional backbone flexibilities provides evidence for a remarkable degree of structural conservation among the proteins surveyed. Parsimony analysis of the protein sequence alignment identifies four monophyletic groups on the arthropodan branch of the hemocyanin gene tree: crustacean hemocyanins, insect hexamerins, chelicerate hemocyanins, and arthropodan prophenoloxidases. They form a monophyletic group relative to molluscan hemocyanins and nonarthropodan tyrosinases. Arthropodan prophenoloxidases, although functionally similar to tyrosinases, appear to belong to the arthropodan hexamer- type hemolymph proteins as opposed to molluscan hemocyanins and tyrosinases.      

Ribostral: an RNA 3D alignment analyzer and viewer based on basepair isostericities

RNA atomic resolution structures have revealed the existance of different families of basepair interactions, each of which with its own isosteric sub-families. Ribostral (Ribonucleic Structural Aligner) is a user-friendly framework for analyzing, evaluating and viewing RNA sequence alignments with at least one available atomic resolution structure. It is the first of its kind that makes direct and easy- to-understand superposition of the isostericity matrices of basepairs observed in the structure onto sequence alignments, easily indicating allowed and unallowed substitutions at each BP position. Potential mistakes in the alignments can then be corrected using other sequence editing software. Ribostral has been developed and tested under Windows XP, and is capable of running on any PC or MAC platform with MATLAB 7.1 (SP3) or higher installed version. A stand-alone version is also available for the PC platform. AVAILABILITY: http://rna.bgsu.edu/ribostral.     

Long-residency hydration, cation binding, and dynamics of loop E/helix IV rRNA-L25 protein complex

Molecular dynamics simulations of RNA-protein complex between Escherichia coli loop E/helix IV (LE/HeIV) rRNA and L25 protein reveal a qualitative agreement between the experimental and simulated structures. The major groove of LE is a prominent rRNA cation-binding site. Divalent cations rigidify the LE major groove geometry whereas in the absence of divalent cations LE extensively interacts with monovalent cations via inner-shell binding. The HeIV region shows bistability of its major groove explaining the observed differences between x-ray and NMR structures. In agreement with the experiments, the simulations suggest that helix-alpha1 of L25 is the least stable part of the protein. Inclusion of Mg2+ cations into the simulations causes perturbation of basepairing at the LE/HeIV junction, which does not, however, affect the protein binding. The rRNA-protein complex is mediated by a number of highly specific hydration sites with long-residing water molecules and two of them are bound throughout the entire 24-ns simulation. Long-residing water molecules are seen also outside the RNA-protein contact areas with water-binding times substantially enhanced compared to simulations of free RNA. Long-residency hydration sites thus represent important elements of the three-dimensional structure of rRNA.     

Non-Watson-Crick basepairing and hydration in RNA motifs: molecular dynamics of 5S rRNA loop E

Explicit solvent and counterion molecular dynamics simulations have been carried out for a total of >80 ns on the bacterial and spinach chloroplast 5S rRNA Loop E motifs. The Loop E sequences form unique duplex architectures composed of seven consecutive non-Watson-Crick basepairs. The starting structure of spinach chloroplast Loop E was modeled using isostericity principles, and the simulations refined the geometries of the three non-Watson-Crick basepairs that differ from the consensus bacterial sequence. The deep groove of Loop E motifs provides unique sites for cation binding. Binding of Mg(2+) rigidifies Loop E and stabilizes its major groove at an intermediate width. In the absence of Mg(2+), the Loop E motifs show an unprecedented degree of inner-shell binding of monovalent cations that, in contrast to Mg(2+), penetrate into the most negative regions inside the deep groove. The spinach chloroplast Loop E shows a marked tendency to compress its deep groove compared with the bacterial consensus. Structures with a narrow deep groove essentially collapse around a string of Na(+) cations with long coordination times. The Loop E non-Watson-Crick basepairing is complemented by highly specific hydration sites ranging from water bridges to hydration pockets hosting 2 to 3 long-residing waters. The ordered hydration is intimately connected with RNA local conformational variations.     

The annotation of RNA motifs

The recent deluge of new RNA structures, including complete atomic-resolution views of both subunits of the ribosome, has on the one hand literally overwhelmed our individual abilities to comprehend the diversity of RNA structure, and on the other hand presented us with new opportunities for comprehensive use of RNA sequences for comparative genetic, evolutionary and phylogenetic studies. Two concepts are key to understanding RNA structure: hierarchical organization of global structure and isostericity of local interactions. Global structure changes extremely slowly, as it relies on conserved long-range tertiary interactions. Tertiary RNA-RNA and quaternary RNA-protein interactions are mediated by RNA motifs, defined as recurrent and ordered arrays of non-Watson-Crick base-pairs. A single RNA motif comprises a family of sequences, all of which can fold into the same three-dimensional structure and can mediate the same interaction(s). The chemistry and geometry of base pairing constrain the evolution of motifs in such a way that random mutations that occur within motifs are accepted or rejected insofar as they can mediate a similar ordered array of interactions. The steps involved in the analysis and annotation of RNA motifs in 3D structures are: (a) decomposition of each motif into non-Watson-Crick base-pairs; (b) geometric classification of each basepair; (c) identification of isosteric substitutions for each basepair by comparison to isostericity matrices; (d) alignment of homologous sequences using the isostericity matrices to identify corresponding positions in the crystal structure; (e) acceptance or rejection of the null hypothesis that the motif is conserved.