Effect of spermidine on the conformation of bacteriophage MS2 RNA. Electron microscopy and computer modeling

The structure of single-stranded RNA from the bacteriophage MS2 has been examined by electron microscopy in the presence of the polyamine spermidine. The molecules are found in two alternate conformations. The first of these can be characterized as a cruciform structure composed of three large loops approximately 500 to 700 nucleotides in size. The interior of the molecule has extensive base-paired regions which connect distant regions of the molecule; the farthest being 2500 nucleotides apart. In the second conformation, the molecules appear rod-like. Two of the large loops disappear, and these regions form, instead, extensive long-range helices. Computer modeling has been employed to explore the base-pairing potential of the sequence of bacteriophage MS2 RNA. Double-stranded regions identified by electron microscopy are shown to occur in local G + C-rich stretches of the RNA. Detailed models have been calculated for two regions of long-range contact. One of these includes the ribosome-binding site for the viral coat protein gene. The results are discussed in the context of the known role of RNA structure in the regulation of viral gene expression.     

Phosphorothioate substitution can substantially alter RNA conformation

Phosphorothioate substitution-interference experiments, routinely used to stereospecifically identify phosphoryl oxygen sites that participate in RNA-ligand binding and RNA-directed catalysis, rest in their interpretation on the untested assumption that substitution does not alter the conformation of the modified molecule from its biologically active state. Using NMR spectroscopy, we have tested this assumption by determining the structural effect of stereospecific phosphorothioate substitution at five positions in an RNA hairpin containing the binding site for bacteriophage MS2 capsid protein. At most sites, substitution has little or no effect, causing minor perturbations in the phosphate backbone and increasing the stacking among nucleotides in the hairpin loop. At one site, however, phosphorothioate substitution causes an unpaired adenine necessary for formation of the capsid protein-RNA complex to loop out of the RNA helix into the major groove. These results indicate that phosphorothioate substitution can substantially alter the conformation of RNA at positions of irregular secondary structure, complicating the use of substitution-interference experiments to study RNA structure and function.     

Structural studies on site-directed mutants of domain 3 of Xenopus laevis oocyte 5 S ribosomal RNA

Base substitutions have been introduced into the highly conserved sequences of loops D and E within domain 3 of Xenopus laevis oocyte 5 S rRNA. The effects of these mutations on the solution structure of this 5 S rRNA have been studied by means of probing with nucleases, and with chemical reagents under native and semi-denaturing conditions. The data obtained with these mutants support the graphic model of Xenopus oocyte 5 S rRNA proposed by Westhof et al. In particular, our results rule out the existence of long-range base-pairing interactions between loop C and either loop D or loop E. The data also confirm that loops D and E in the wild-type 5 S RNA adopt unusual secondary structures and illustrate the importance of nucleotide sequence in the formation of intrinsic local loop conformations via non-canonical base-pairs and specific base-phosphate contacts. Consistent with this conclusion is our observation that the domain 3 fragment of Xenopus oocyte 5 S rRNA adopts the same conformation as the corresponding region in the full-length 5 S rRNA.     

Interactions of cations with RNA loop-loop complexes

RNA loop-loop interactions are essential in many biological processes, including initiation of RNA folding into complex tertiary shapes, promotion of dimerization, and viral replication. In this article, we examine interactions of metal ions with five RNA loop-loop complexes of unique biological significance using explicit-solvent molecular-dynamics simulations. These simulations revealed the presence of solvent-accessible tunnels through the major groove of loop-loop interactions that attract and retain cations. Ion dynamics inside these loop-loop complexes were distinctly different from the dynamics of the counterion cloud surrounding RNA and depend on the number of basepairs between loops, purine sequence symmetry, and presence of unpaired nucleotides. The cationic uptake by kissing loops depends on the number of basepairs between loops. It is interesting that loop-loop complexes with similar functionality showed similarities in cation dynamics despite differences in sequence and loop size.     

Experimentally derived nearest-neighbor parameters for the stability of RNA three- and four-way multibranch loops.

Algorithms for predicting RNA secondary structure require approximations for the free energies of multibranch loops, also called junctions. The stabilities of 62 RNA duplexes with three- and four-way multibranch loops were determined by optical melting. To account for the observed sequence dependence, a revised loop free-energy approximation is proposed that accounts for the strain in three-way junctions with fewer than two unpaired nucleotides, penalizes asymmetry in the distribution of unpaired nucleotides, and gives a bonus for four-way loops relative to three-way loops. Parameters for this equation were determined by linear regression.     

Sequence dependence for the energetics of dangling ends and terminal base pairs in ribonucleic acid

Stability increments of terminal unpaired nucleotides (dangling ends) and terminal base pairs on the core helixes AUGCAU and UGCGCA are reported. Enthalpy, entropy, and free energy changes of helix formation were measured spectrophotometrically for 18 oligoribonucleotides containing the core sequences. The results indicate 3' dangling purines add more stability than 3' dangling pyrimidines. In most cases, the additional stability from a 3' dangling end on an AU base pair is less than that on a GC base pair [Freier, S.M., Burger, B.J., Alkema, D., Neilson, T., & Turner, D.H. (1985) Biochemistry 22, 6198-6206]. The sequence dependence provides a test for the importance of dangling ends for various RNA interactions. Correlations are suggested with codon context effects and with the three-dimensional structure of yeast phenylalanine transfer RNA. In the latter case, all terminal unpaired nucleotides having stability increments more favorable than -1 kcal/mol are stacked on the adjacent base pair. All terminal unpaired nucleotides having stability increments less favorable than -0.3 kcal/mol are not stacked on the adjacent base pair. In several cases, this lack of stacking is associated with a turn in the sugar-phosphate backbone. This suggests stability increments measured on oligoribonucleotides may be useful for predicting tertiary structure in large RNA molecules. Comparison of the stability increments for terminal dangling ends and base pairs, and of terminal GC and AU base pairs, indicates the free energy increment associated with forming a hydrogen bond can be about -1 kcal/mol of hydrogen bond.     

Structure of 4.5S RNA in the signal recognition particle of Escherichia coli as studied by enzymatic and chemical probing.

The structure of 4.5S RNA, the Escherichia coli homologue of the signal recognition particle (SRP) RNA, alone and in the SRP complex with protein P48 (Ffh) was probed both enzymatically and chemically. The molecule is largely resistant against single strand-specific nucleases, indicating a highly base paired structure. Reactivity appears mainly in the apical tetraloop and in one of the conserved internal loops. Although some residues are found reactive toward dimethylsulphate and kethoxal in regions predicted to be unpaired by the phylogenetic secondary structure model of 4.5S RNA, generally the reactivity is low, and some residues in internal loops are not reactive at all. RNase V1 cleaves the RNA at multiple sites that coincide with predicted helices, although the cleavages show a pronounced asymmetry. The binding of protein P48 to 4.5S RNA results in a protection of residues in the apical part of the molecule homologous to eukaryotic SRP RNA (domain IV), whereas the cleavages in the conserved apical tetraloop are not protected. Hydroxyl radical treatment reveals an asymmetric pattern of backbone reactivity; in particular, the region encompassing nucleotides 60-82, i.e., the 3' part of the conserved domain IV, is protected. The data suggest that a bend in the domain IV region, most likely at the central asymmetric internal loop, is an important element of the tertiary structure of 4.5S RNA. Hyperchromicity and lead cleavage data are consistent with the model as they reveal the unfolding of a higher-order structure between 30 and 40 degrees C. Protection by protein P48 occurs in this region of the RNA and, more strongly, in the 5' part of domain IV (nt 26-50, most strongly from 35 to 49). It is likely that P48 binds to the outside of the bent form of 4.5S RNA.     

Higher order structure of chloroplastic 5S ribosomal RNA from spinach

The secondary and tertiary structure of chloroplastic 5S ribosomal RNA from spinach was investigated by the use of several chemical and enzymatic structure probes. The four bases were monitored at one of their Watson-Crick base-pairing positions with dimethyl sulfate [at A(N1) and C(N3)] and with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate [at G(N1) and U(N3)]. Position N7 of purines was probed with diethyl pyrocarbonate (adenines) and with dimethyl sulfate (guanines). Ethylnitrosourea was used to probe phosphate involved in tertiary interaction or in cation coordination. In order to estimate the degree of stability of helices, the various chemical reagents were employed under "native" conditions (300 mM KCl and 20 mM magnesium at 37 degrees C), under "semidenaturing" conditions [1 mM ethylenediaminetetraacetic acid (EDTA) at 37 degrees C], and under denaturing conditions (1 mM EDTA at 90 degrees C). Unstructured regions were also tested with single-strand-specific nucleases T1, U2, and S1 and double-stranded or stacked regions with RNase V1 from cobra Naja naja oxiana venom. The results confirm the existence of the five helices and the two external loops proposed in the consensus model of 5S rRNA. However, the regions depicted as unpaired internal loops appear to be folded into a more complex conformation. A three-dimensional model derived from the present data and graphic modeling for a region encompassing helix IV, helix V, loop D, and loop E (nucleotides 70-110) is proposed. Nucleotides in the so-called loop E (73-79/100-106) display unusual features: Noncanonical base pairs (A-A and A-G) are formed, and three nucleotides (C75, U78, and U105) are bulging out. This region adopts an unwound and extended conformation that can be well suited for tertiary interactions or for protein binding. Several bases and phosphates candidate for the tertiary folding of the RNA were also identified.     

The conserved 3′X terminal domain of hepatitis C virus genomic RNA forms a two-stem structure that promotes viral RNA dimerization

The 3′X domain of hepatitis C virus is a strongly conserved structure located at the 3′ terminus of the viral genomic RNA. This domain modulates the replication and translation processes of the virus in conjunction with an upstream 5BSL3.2 stem–loop, and contains a palindromic sequence that facilitates RNA dimerization. Based on nuclear magnetic resonance spectroscopy and gel electrophoresis, we report here that domain 3′X adopts a structure composed of two stem–loops, and not three hairpins or a mixture of folds, as previously proposed. This structure exposes unpaired terminal nucleotides after a double-helical stem and palindromic bases in an apical loop, favoring genomic RNA replication and self-association. At higher ionic strength the domain forms homodimers comprising an intermolecular duplex of 110 nucleotides. The 3′X sequences can alternatively form heterodimers with 5BSL3.2. This contact, reported to favor translation, likely involves local melting of one of the 3′X stem–loops.     

Solution structure of an alternate conformation of helix27 from Escherichia coli16S rRNA

Helix (H)27 of 16S ribosomal (r)RNA from Escherichia coli was dubbed the "switch helix" when mutagenesis suggested that two alternative base pair registers may have distinct functional roles in the bacterial ribosome. Although more recent genetic analyses suggest that H27 conformational switching is not required for translation, previous solution studies demonstrated that the isolated E. coli H27 can dynamically convert between the 885 and 888 conformations. Here, we have solved the nuclear magnetic resonance solution structure of a locked 888 conformation. NOE and residual dipolar coupling restraints reveal an architecture that markedly differs from that of the 885 conformation found in crystal structures of the bacterial ribosome. In place of the loop E motif that characterizes the 885 conformer and that the 888 conformer cannot adopt, we find evidence for an asymmetrical A-rich internal loop stabilized by stacking interactions among the unpaired A's. Comparison of the isolated H27 888 solution structure with the 885 crystal structure within the context of the ribosome suggests a difference in overall length of H27 that presents one plausible reason for the absence of H27 conformational switching within the sterically confining ribosome.     

UNAC tetraloops: to what extent do they mimic GNRA tetraloops?

The structures of four small RNAs each containing a different version of the UNAC loop were determined in solution using NMR spectroscopy and restrained molecular dynamics. The UMAC tetraloops (where M is A or C) exhibited a typical GNRA fold including at least one hydrogen bond between the first U and fourth C. In contrast, UGAC and UUAC tetraloops have a different orientation of the first and fourth residues, such that they do not closely mimic the GNRA fold. Although the UMAC tetraloops are excellent structural mimics of the GNRA tetraloop backbone, sequence comparisons typically do not reveal co‐variation between the two loop types. The limited covariation is attributed to differences in the location of potential hydrogen bond donors and acceptors as a result of the replacement of the terminal A of GNRA with C in the UMAC version. Thus, UMAC loops do not readily form the common GNRA tetraloop‐receptor interaction. The loop at positions 863‐866 in E. coli 16S ribosomal RNA appears to be a major exception. However, in this case the GNRA loop does not in fact engage in the usual base to backbone tertiary interactions. In summary, UMAC loops are not just an alternative sequence version of the GNRA loop family, but instead they expand the types of interactions, or lack thereof, that are possible. From a synthetic biology perspective their inclusion in an artificial RNA may allow the establishment of a stable loop structure while minimizing unwanted long range interactions or permitting alternative long‐range interactions. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 617–628, 2012.     

RNase-stable RNA: conformational parameters of the nucleic acid backbone for binding to RNase T1.

An RNA sequence showing high stability with respect to digestion by ribonuclease T1 (RNase T1) was isolated by in vitro selection from an RNA library. Although ribonuclease T1 cleaves single-stranded RNA specifically after guanosine residues, secondary structure calculations predict several guanosines in single-stranded areas. Two of these guanosines are part of a GGCA-tetraloop, a recurring structure element in the secondary structure predictions. Molecular dynamics simulations of the conformation space of the nucleotides involved in this tetraloop show on the one hand that the nucleic acid backbone of the guanosines cannot realise the conformation required for cleavage by RNase T1. On the other hand, it could be shown that an RNA molecule not forced into a tetraloop occupies this conformation several times in the course of the simulation. The simulations confirm the GGCA-tetraloop as an RNase-stable secondary structure element. Our results show that, besides the known prerequisite of a single-stranded RNA, RNase T1 has additional demands on the substrate conformation.     

A new model for the tertiary structure of 5S ribonucleic acid in plants

Chemical, enzymatic and physicochemical methods of a structural analysis of 5S rRNAs in lupine, wheat germ, and other plants led us to propose a new three-dimensional model of these molecules The main features of the model are tertiary interactions between the β- and γ-domains of the molecule, specifically nucleotides (34)CCCA(37) in loop C and nucleotides (85)GGGU(88) in loop D. In addition we propose tertiary base-pairing in A100-U53 between loops B and E. We have confirmed this model by NMR spectroscopy and by chemical modification with diethylpyrocarbonate. Our results are consistent with the proposed model and are also applicable to all eukaryotic 5S rRNAs. Our model is clearly differentiated from others by intramolecular tertiary hydrogen bonds between the two domains.     

Dissecting the protein-RNA interface: the role of protein surface shapes and RNA secondary structures in protein-RNA recognition

Protein-RNA interactions are essential for many biological processes. However, the structural mechanisms underlying these interactions are not fully understood. Here, we analyzed the protein surface shape (dented, intermediate or protruded) and the RNA base pairing properties (paired or unpaired nucleotides) at the interfaces of 91 protein-RNA complexes derived from the Protein Data Bank. Dented protein surfaces prefer unpaired nucleotides to paired ones at the interface, and hydrogen bonds frequently occur between the protein backbone and RNA bases. In contrast, protruded protein surfaces do not show such a preference, rather, electrostatic interactions initiate the formation of hydrogen bonds between positively charged amino acids and RNA phosphate groups. Interestingly, in many protein-RNA complexes that interact via an RNA loop, an aspartic acid is favored at the interface. Moreover, in most of these complexes, nucleotide bases in the RNA loop are flipped out and form hydrogen bonds with the protein, which suggests that aspartic acid is important for RNA loop recognition through a base-flipping process. This study provides fundamental insights into the role of the shape of the protein surface and RNA secondary structures in mediating protein-RNA interactions.     

Structure of an AAGU tetraloop and its contribution to substrate selection by yeast RNase III

RNase III enzymes are a highly conserved family of proteins that specifically cleave double-stranded RNA (dsRNA). These proteins are involved in a variety of cellular functions, including the processing of many non-coding RNAs, mRNA decay, and RNA interference. In yeast Rnt1p, a dsRNA-binding domain (dsRBD) recognizes its substrate by interacting with stems capped with conserved AGNN tetraloops. The enzyme uses the tetraloop to cut 14nt to 16nt away into the stem in a ruler-like mechanism. The solution structure of Rnt1p dsRBD complexed to one of its small nucleolar (sno) RNA substrate revealed non-sequence-specific contacts with the sugar-phosphate backbone in the minor groove of the AGNN fold and the two non-conserved tetraloop nucleotides. Recently, a new form of Rnt1p substrates lacking the conserved AGNN sequence but instead harboring an AAGU tetraloop was found at the 5' end of snoRNA 48 precursor. Here, we report the solution structure of this hairpin capped with an AAGU tetraloop. Some of the stacking interactions and the position of the turn in the sugar-phosphate backbone are similar to the one observed in the AGNN loop structure; however, the AAGU sequence adopts a different conformation. The most striking difference was found at the 3' end of the loop where Rnt1p interacts with AGNN substrates. The last nucleotide is extruded from the AAGU tetraloop structure in contrast to the compact AGNN fold. The AAGU hairpin structure suggests that Rnt1p recognizes substrates with different tetraloop structures, indicating that the structural repertoire specifically recognized by Rnt1p is larger than previously anticipated.     

Fluorescence competition and optical melting measurements of RNA three-way multibranch loops provide a revised model for thermodynamic parameters

Three-way multibranch loops (junctions) are common in RNA secondary structures. Computer algorithms such as RNAstructure and MFOLD do not consider the identity of unpaired nucleotides in multibranch loops when predicting secondary structure. There is limited experimental data, however, to parametrize this aspect of these algorithms. In this study, UV optical melting and a fluorescence competition assay are used to measure stabilities of multibranch loops containing up to five unpaired adenosines or uridines or a loop E motif. These results provide a test of our understanding of the factors affecting multibranch loop stability and provide revised parameters for predicting stability. The results should help to improve predictions of RNA secondary structure.