Structural features that give rise to the unusual stability of RNA hairpins containing GNRA loops.

The most frequently occurring RNA hairpins in 16S and 23S ribosomal RNA contain a tetranucleotide loop that has a GNRA consensus sequence. The solution structures of the GCAA and GAAA hairpins have been determined by nuclear magnetic resonance spectroscopy. Both loops contain an unusual G-A base pair between the first and last residue in the loop, a hydrogen bond between a G base and a phosphate, extensive base stacking, and a hydrogen bond between a sugar 2'-end OH and a base. These interactions explain the high stability of these hairpins and the sequence requirements for the variant and invariant nucleotides in the GNRA tetranucleotide loop family.

NMR structure of a classical pseudoknot: interplay of single- and double-stranded RNA.

Pseudoknot formation folds the 3' ends of many plant viral genomic RNAs into structures that resemble transfer RNA in global folding and in their reactivity to transfer RNA-specific proteins. The solution structure of the pseudoknotted T arm and acceptor arm of the transfer RNA-like structure of turnip yellow mosaic virus (TYMV) was determined by nuclear magnetic resonance (NMR) spectroscopy. The molecule is stabilized by the hairpin formed by the 5' end of the RNA, and by the intricate interactions related to the loops of the pseudoknot. Loop 1 spans the major groove of the helix with only two of its four nucleotides. Loop 2, which crosses the minor groove, interacts closely with its opposing helix, in particular through hydrogen bonds with a highly conserved adenine. The structure resulting from this interaction between the minor groove and single-stranded RNA at helical junctions displays internal mobility, which may be a general feature of RNA pseudoknots that regulates their interaction with proteins or other RNA molecules.

Is there a special function for U.G basepairs in ribosomal RNA?

U.G basepairs are well-established elements of RNA structure. The geometry of this pair is different, however, from classical Watson-Crick basepairs. This leads to an unusual stacking of the basepair: overlap with the basepair at the 5' side of the U (and the 3' side of the G) is strong (stacked) while it is weak with the basepair on the other side (destacked). The closure of an RNA helix by a U.G pair will be energetically unfavourable when the U residue occupies the 5' end. In transfer RNA there is a strong selection against a 'destacked' U.G pair at helix ends. In the 16S rRNA model of Escherichia coli there are 72 U.G pairs of which 36 or 22 occupy a helix end, depending on how such an end is defined. There is a slight preference for 'stacked' U.G's in these positions. It is remarkable, however, that of 13 very conserved U.G pairs in the 16S (-like) rRNA, 7 occur at helix ends and that 5 of these have the 'destacked' configuration. It is suggested that these pairs, if they exist at all in a hydrogen-bounded form, are stabilized by co-axial stacking with other helices or by interaction with a protein.

Nuclear magnetic resonance studies of the hammerhead ribozyme domain. Secondary structure formation and magnesium ion dependence.

Proton nuclear magnetic resonance (n.m.r.) experiments were used to probe base-pair formation in several hammerhead RNA enzyme (ribozyme) domains. The hammerhead domains consist of a 34 nucleotide ribozyme bound to a complementary 13 nucleotide non-cleavable DNA substrate. Three hammerhead domains were studied that differ in the sequence and stability of one of the helices involved in recognition of the substrate by the ribozyme. The n.m.r. data show a 1:1 stoichiometry for the ribozyme-substrate complexes. The imino proton resonances in the hammerhead complexes were assigned by two-dimensional nuclear Overhauser effect experiments. These data confirm the presence of two of the three helical regions in the hammerhead domain, predicted from phylogenetic data; and are also consistent with the formation of the third helix. Since a divalent cation is required for efficient catalytic activity of the hammerhead domain, the magnesium ion dependence of the n.m.r. spectra was studied for two of the hammerhead complexes. One of the complexes showed very large spectral changes upon addition of magnesium ions. However, the complex that has the most C.G base-pairs in one of the recognition helices shows essentially no spectral (and therefore presumably structural) changes upon addition of magnesium. These data are consistent with a model where the magnesium binding site already exists in the magnesium-free complex, suggesting that the magnesium ion serves primarily a catalytic, and not a structural, role under the conditions used here.

The detailed structure of tandem G.A mismatched base-pair motifs in RNA duplexes is context dependent.

The solution structure of the RNA duplex (rGGGCUGAAGCCCU), containing tandem G.A mismatches has been determined by NMR spectroscopy and restrained molecular dynamics. A homonuclear 3D TOCSY-NOESY was used to derive 18 to 30 distance restraints per nucleotide, as well as all gamma torsion angles and sugar puckers for the central UGAA part of the molecule. Using these constraints, together with cross-strand distances, involving exchangeable imino protons, and essentially all other torsion angles that can accurately be determined (i.e. beta, epsilon) otherwise, the structure of the UGAA domain could be determined with high precision (r.m.s.d. 0.62 A), without the aid of isotopically enriched RNA. The G.A base-pairs are of the sheared pairing type, with both nucleotides in the anti conformation, and hydrogen bonds between the guanine 2-amino and the adenine N7 and between the guanine N3 and the adenine 6-amino. Surprisingly the sugar of the guanosine of the G.A. mismatch adopts a 2'-endo sugar pucker conformation. Comparison with other RNA structures, in which two such G.A base-pairs are formed reveals that this detailed structure depends on the identity of the base 5' to the guanosine in the tandem G.A base-pairs. A geometrical model for the incorporation of sheared tandem G.A base-pairs in A-form helices is formulated, which explains the distinct different stacking properties and helical parameters in sequences containing tandem, sheared G.A base-pairs.

A network of heterogeneous hydrogen bonds in GNRA tetraloops.

RNA hairpin loops containing a GNRA consensus sequence are the most frequently occurring hairpins in a variety of prokaryotic and eukaryotic RNAs. These tetraloops play important functional roles in RNA folding, in RNA-RNA tertiary interactions and as protein binding sites. Homo and heteronuclear NMR spectroscopy have been used to determine the structures of the most abundant members of the GNRA tetraloop family: the GAGA, GCAA and GAAA loops closed by a C-G base pair. Analysis of the structures of these three hairpin loops reveals a network of heterogeneous hydrogen bonds. The loops contain a G-A base pair, a G base-phosphate hydrogen bond and several 2' OH-base hydrogen bonds. These intramolecular interactions and the extensive base stacking in the loop help explain the high thermodynamic stability and give insight into the diverse biological roles of the GNRA RNA hairpins.

Solution structure of the pseudoknot of SRV-1 RNA, involved in ribosomal frameshifting.

RNA pseudoknots play important roles in many biological processes. In the simian retrovirus type-1 (SRV-1) a pseudoknot together with a heptanucleotide slippery sequence are responsible for programmed ribosomal frameshifting, a translational recoding mechanism used to control expression of the Gag-Pol polyprotein from overlapping gag and pol open reading frames. Here we present the three-dimensional structure of the SRV-1 pseudoknot determined by NMR. The structure has a classical H-type fold and forms a triple helix by interactions between loop 2 and the minor groove of stem 1 involving base-base and base-sugar interactions and a ribose zipper motif, not identified in pseudoknots so far. Further stabilization is provided by a stack of five adenine bases and a uracil in loop 2, enforcing a cytidine to bulge. The two stems of the pseudoknot stack upon each other, demonstrating that a pseudoknot without an intercalated base at the junction can induce efficient frameshifting. Results of mutagenesis data are explained in context with the present three-dimensional structure. The two base-pairs at the junction of stem 1 and 2 have a helical twist of approximately 49 degrees, allowing proper alignment and close approach of the three different strands at the junction. In addition to the overwound junction the structure is somewhat kinked between stem 1 and 2, assisting the single adenosine in spanning the major groove of stem 2. Geometrical models are presented that reveal the importance of the magnitude of the helical twist at the junction in determining the overall architecture of classical pseudoknots, in particular related to the opening of the minor groove of stem 1 and the orientation of stem 2, which determines the number of loop 1 nucleotides that span its major groove.

High-resolution proton magnetic resonance studies of the 3'-terminal colicin fragment of 16 S ribosomal RNA from Escherichia coli. Assignment of iminoproton resonances by nuclear Overhauser effect experiments and the influence of adenine dimethylation on the hairpin conformation.

The "colicin" fragments comprising the 49 3'-terminal nucleotides of 16 S ribosomal RNA have been isolated from wild-type Escherichia coli and from a kasugamycin-resistant mutant that lacks methylation of two geminal adenine residues. Proton nuclear magnetic resonance (n.m.r.) spectra (500 MHz) were recorded at various temperatures. The low-field resonances arising from the hydrogen-bonded iminoprotons of paired bases were assigned using the nuclear Overhauser effect (n.o.e.). Crucial to the interpretation of the spectra are the resonances that originate from the two hydrogen-bonded iminoprotons of a U X G basepair. Combined with temperature-jump relaxation kinetics experiments the n.o.e.s lead to the conclusion that a conserved A X U/U X G junction in the hairpin is a thermolabile dislocation in the helix. The n.m.r. spectra of the wild-type and mutant fragment are only different with respect to the iminoproton resonances of the two base-pairs adjoining the hairpin loop. The spectra recorded at various temperatures tend to indicate that dimethylation of the adenosines labilizes these base-pairs, but no definitive conclusions are drawn. The results confirm our previous views that dimethylation of the adenosine residues affects the conformation of the hairpin loop.

Solution structure of a HNA-RNA hybrid.

BACKGROUND: Synthetic nucleic acid analogues with a conformationally restricted sugar-phosphate backbone are widely used in antisense strategies for biomedical and biochemical applications. The modified backbone protects the oligonucleotides against degradation within the living cell, which allows them to form stable duplexes with sequences in target mRNAs with the aim of arresting their translation. The biologically most active antisense oligonucleotides also trigger cleavage of the target RNA through activation of endogenous RNase H. Systematic studies of synthetic oligonucleotides have also been conducted to delineate the origin of the chirality of DNA and RNA that are both composed of D-nucleosides. RESULTS: Hexitol nucleic acids (HNA) are the first example of oligonucleotides with a six-membered carbohydrate moiety that can bind strongly and selectively to complementary RNA oligomers. We present the first high resolution nuclear magnetic resonance structure of a HNA oligomer bound to a complementary RNA strand. The HNA-RNA complex forms an anti-parallel heteroduplex and adopts a helical conformation that belongs to the A-type family. Possibly, due to the rigidity of the rigid chair conformation of the six-membered ring both the HNA and RNA strand in the duplex are well defined. The observed absence of end-fraying effects also indicate a reduced conformational flexibility of the HNA-RNA duplex compared to canonical dsRNA or an RNA-DNA duplex. CONCLUSIONS: The P-P distance across the minor groove, which is close to A-form, and the rigid conformation of the HNA-RNA complex, explain its resistance towards degradation by Rnase H. The A-form character of the HNA-RNA duplex and the reduced flexibility of the HNA strand is possibly responsible for the stereoselectivity of HNA templates in non-enzymatic replication of oligonucleotides, supporting the theory that nucleosides with six-membered rings could have existed at some stage in molecular evolution.

The conformation of a conserved stem-loop structure in ribosomal RNA.

The RNA of small ribosomal subunits contains a conserved stem-loop structure near the 3' end. Characteristics for the hairpins are: (a) a nine-basepairs stem: (b) a conserved A-UU-G junction in the stem: (c) a conserved sequence Gm6(2)AM6(2)A sequence in the loop (except yeast mitochondria and mutants from bacteria). We are using UV-optics, micro-calorimetry and 500 MHz-NMR to investigate fragments of about 50 nucleotides cleaved from the 3' ends of small ribosomal subunit RNA's by bacteriocins. Our preliminary conclusions are: (1) Dimethylation of the adenines in the loop destabilizes the hairpin because of an increased stacking; (2) melting of the hairpin starts at the ends as well as in the middle at the A-UU-G junction; (3) basepair substitutions have an unexpectedly large effect on thermal stability.

The 3' terminal colicin fragment of Escherichia coli 16S ribosomal RNA. Conformational details revealed by enzymic and chemical probing.

The conformation of the colicin fragment of E. coli 16S rRNA was probed with various nucleases and with the adenosine-specific reagent diethylpyrocarbonate (DEP). The results confirm the presence of a stable central hairpin in the colicin fragment and a weaker additional secondary structure involving the regions 5' and 3' to this hairpin. By monitoring DEP accessibility at various stages of heat-denaturation sequential unfolding of individual base pairs was followed. In agreement with previous results it could be shown that dimethylation of the two adjacent adenosines in the hairpin loop (a feature in virtually all ribosomes) leads to a destabilization of the hairpin helix. Accessibilities of G residues, involved in the weaker additional secondary structure is anomalous. One G residue is sensitive to the single strand specific RNase T1 and insensitive to DEP, while the situation is reversed for the adjoining G residue. The strong reaction of the latter G-residue with DEP is unusual and indicates a very special conformation.

Genetics of limb development and congenital hand malformations.

The vertebrate limb bud develops along three different axes: proximodistal, anteroposterior, and dorsoventral. Several genetic factors responsible for control of each of the three limb axes have been identified. The genes involved interact in complex feedback loops to achieve proper arrangement and differentiation of tissues. Most of the available information on limb development and patterning has come from studies carried out in the lower vertebrates. In recent years, an increasing number of studies have been unraveling the genetic basis of human hand malformation phenotypes. At present, genes responsible for preaxial polydactyly, split hand/split foot malformation, and brachydactyly type C have been localized, and the gene responsible for synpolydactyly has been identified. In this paper, we present an overview of the genetic factors involved in limb development, followed by summarized discoveries in the genetics of human congenital hand malformations.

DNA-functionalized hydrogels for confined membrane-free in vitro transcription/translation.

We microfluidically fabricate bio-orthogonal DNA-functionalized porous hydrogels from hyaluronic acid that are employed in in vitro transcription/translation (IVTT) of a green fluorescent protein. By co-encapsulating individual hydrogel particles and the IVTT machinery in water-in-oil microdroplets, we study protein expression in a defined reaction volume. Our approach enables precise control over protein expression rates by gene dosage. We show that gene transcription and translation are confined to the membrane-free hydrogel matrix, which contributes to the design of membrane-free protocells.

The structure of the isolated, central hairpin of the HDV antigenomic ribozyme: novel structural features and similarity of the loop in the ribozyme and free in solution.

The structure of an RNA hairpin containing a seven-nucleotide loop that is present in the self-cleaving sequence of hepatitis delta virus antigenomic RNA was determined by high resolution NMR spectroscopy. The loop, which is composed of only one purine and six pyrimidines, has a suprisingly stable structure, mainly supported by sugar hydroxyl hydrogen bonds and base-base and base-phosphate stacking interactions. Compared with the structurally well-determined, seven-membered anticodon loop in tRNA, the sharp turn which affects the required 180 degrees change in direction of the sugar-phosphate backbone in the loop is shifted one nucleotide in the 3' direction. This change in direction can be characterized as a reversed U-turn. It is expected that the reversed U-turn may be found frequently in other molecules as well. There is evidence for a new non-Watson-Crick UC base pair formed between the first and the last residue in the loop, while most of the other bases in the loop are pointing outwards making them accessible to solvent. From chemical modification, mutational and photocrosslinking studies, a similar picture develops for the structure of the hairpin in the active ribozyme indicating that the loop structure in the isolated hairpin and in the ribozyme is very similar.

New developments in structure determination of pseudoknots.

Recently, several high-resolution structures of-RNA pseudoknots have become available. Here we review the progress in this area. The majority of the structures obtained belong to the classical or H-type pseudoknot family. The most complicated pseudoknot structure elucidated so far is the Hepatitis Delta Virus ribozyme, which forms a nested double pseudoknot. In particular, the structure-function relationships of the H-type pseudoknots involved in translational frameshifting have received much attention. All molecules considered show interesting new structural motifs.

Sequence-dependent structural variations of hammerhead RNA enzymes.

The discovery of in vivo catalytic activity for the hammerhead RNA self-cleaving domain has led to the development of a new class of sequence-specific RNA endonucleases. Two such ribozymes have been synthesized using in vitro transcription with T7 polymerase and their structures have been studied by optical spectroscopy, nuclear magnetic resonance and nondenaturing gel electrophoresis. These data show the presence of a stable hairpin consisting of a double helical stem and a tetranucleotide loop in both RNA enzymes. Additional structure, with different stabilities, is also observed in both RNA enzymes. The half-lives for cleavage of the complementary RNA substrates by these two RNA enzymes have been previously shown to differ by a factor of 50. The data presented here suggest that this rate difference may be a result of the formation of catalytically inactive conformations in the RNA enzyme which interfere with formation of the enzyme-substrate complex.

16S ribosomal RNA of Escherichia coli contains a N2-methylguanosine at 27 nucleotides from the 3' end.

The 49 nucleotides fragment derived from the 3' end of 16S rRNA by cloacin DF13, is not cleaved by ribonuclease T1 at a guanosine residue tha is present at 27 nucleotides from the 3' terminus (position 115 in 16S rRNA). Analysis of the isolated nucleotide indicates that it is a modified G residue. In vivo labeling with (3H)methionine shows that this G is methylated and co-chromatography with markers reveals that it is N2-methylguanosine.

Circular dichroism and 500-MHz proton magnetic resonance studies of the interaction of Escherichia coli translational initiation factor 3 protein with the 16S ribosomal RNA 3' cloacin fragment.

The RNA helix destabilizing properties of Escherichia coli initiation factor 3 protein (IF3), and its affinity for an evolutionarily conserved sequence at the 3' end of 16S rRNA, led us to examine the details of the protein-nucleic acid interactions upon IF3 binding to the 49-nucleotide 3'-terminal cloacin DF13 fragment of 16S rRNA by studying the circular dichroism (CD) and proton magnetic resonance spectra of the RNA, the protein, and their complex. In a physiological tris(hydroxymethyl)aminomethane buffer, where the interaction is primarily nonionic and sequence specific, addition of IF3 decreases the RNA 268-nm CD peak hyperbolically by 19% to an end point of about one IF3 per RNA strand. The titration curve is best fit by an association constant of (1.80 +/- 0.05) X 10(7) M-1, within the range estimated by a nuclease mapping study of the same system [Wickstrom, E. (1983) Nucleic Acids Res. 11, 2035-2052]. In a low-salt phosphate buffer without Mg2+, where the interaction is primarily ionic and nonspecific, titration with IF3 decreases the peak CD sigmoidally by 35% to an end point of two IF3 per strand. The titration curve is best fit by an intrinsic association constant of (1.7 +/- 0.7) X 10(6) M-1 for each IF3 and a cooperativity constant of 33 +/- 6. In a physiological phosphate buffer lacking Mg2+, the dispersion of aromatic proton magnetic resonance peaks and upfield-shifted methyl proton resonances indicates a high degree of secondary and tertiary structure in the protein. In an equimolar mixture of IF3 and RNA cloacin fragment, several changes in identifiable IF3 and RNA resonances are observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Irradiated (15N)DNA as an internal standard for analysis of base-oxidized DNA constituents by isotope dilution mass spectrometry.

A simple and convenient procedure for the preparation of isotopically labeled DNA enriched in oxidized deoxynucleosides is described. 15N-Labeled DNA was isolated from Escherichia coli cells grown in an isotopically enriched medium, and the level of oxidative damage was increased by in vitro irradiation under oxygen. The resulting DNA was hydrolyzed and subsequently analyzed by GC/MS. Results indicated that the DNA was 99% 15N-enriched and that 1% of the total 2'-deoxyguanosine was converted into 8-hydroxy-2'-deoxyguanosine (8-OHdG). When applied to the analysis of 8-OHdG, [15N]DNA as internal standard gave a better reproducibility (CV, 7.9%; n = 5) as compared to the monomeric 8-[18O]hydroxy-2'-deoxyguanosine (CV, 16%; n = 4). Background levels of 8-OHdG in rat colon DNA determined with [15N]DNA and 8-18OHdG as internal standard were 26 +/- 11 and 15 +/- 7 8-OHdG per 10(6) deoxynucleosides, respectively.