Detailed analysis of RNA-protein interactions within the ribosomal protein S8-rRNA complex from the archaeon Methanococcus jannaschii

The crystal structure of ribosomal protein S8 bound to its target 16 S rRNA from a hyperthermophilic archaeon Methanococcus jannaschii has been determined at 2.6 A resolution. The protein interacts with the minor groove of helix H21 at two sites located one helical turn apart, with S8 forming a bridge over the RNA major groove. The specificity of binding is essentially provided by the C-terminal domain of S8 and the highly conserved nucleotide core, characterized by two dinucleotide platforms, facing each other. The first platform (A595-A596), which is the less phylogenetically and structurally constrained, does not directly contact the protein but has an important shaping role in inducing cross-strand stacking interactions. The second platform (U641-A642) is specifically recognized by the protein. The universally conserved A642 plays a pivotal role by ensuring the cohesion of the complex organization of the core through an array of hydrogen bonds, including the G597-C643-U641 base triple. In addition, A642 provides the unique base-specific interaction with the conserved Ser105, while the Thr106 - Thr107 peptide link is stacked on its purine ring. Noteworthy, the specific recognition of this tripeptide (Thr-Ser-Thr/Ser) is parallel to the recognition of an RNA tetraloop by a dinucleotide platform in the P4-P6 ribozyme domain of group I intron. This suggests a general dual role of dinucleotide platforms in recognition of RNA or peptide motifs. One prominent feature is that conserved side-chain amino acids, as well as conserved bases, are essentially involved in maintaining tertiary folds. The specificity of binding is mainly driven by shape complementarity, which is increased by the hydrophobic part of side-chains. The remarkable similarity of this complex with its homologue in the T. thermophilus 30 S subunit indicates a conserved interaction mode between Archaea and Bacteria.     

The RNA binding site of S8 ribosomal protein of Escherichia coli: Selex and hydroxyl radical probing studies.

The RNA binding site of ribosomal protein S8 of Escherichia coli is confined to a small region within the stem of a hairpin in 16S rRNA (nt 588-605/633-651), and thus represents a model system for understanding RNA/protein interaction rules. The S8 binding site on 16S rRNA was suspected to contain noncanonical features difficult to prove with classical genetical or biochemical means. We performed in vitro iterative selection of RNA aptamers that bind S8. For the different aptamers, the interactions with the protein were probed with hydroxyl radicals. Aptamers that were recognized according to the same structural rules as wild-type RNA, but with variations not found in nature, were identified. These aptamers revealed features in the S8 binding site that had been concealed during previous characterizations by the high base conservation throughout evolution. Our data demonstrate that the core structure of the S8 binding site is composed of three interdependent bases (nt 597/641/643), with an essential intervening adenine nucleotide (position 642). The other elements important for the binding site are a base pair (598/640) above the three interdependent bases and a bulged base at position 595, the identity of which is not important. Possible implications on the geometry of the S8 binding site are discussed with the help of a three-dimensional model.     

The crystal structure of the dimerization initiation site of genomic HIV-1 RNA reveals an extended duplex with two adenine bulges

BACKGROUND: An important step in retroviral replication is dimerization of the genomic RNA prior to encapsidation. Dimerization is initiated by the formation of a transient 'kissing-loop complex' that is thought to be subsequently matured into an extended duplex by the nucleocapsid protein (NCp). Although chemical probing and nuclear magnetic resonance spectroscopy have provided insight into the structure of the kissing-loop structure, no structural information concerning the extended-duplex state is available so far. RESULTS: The structure of a minimal HIV-1 RNA dimerization initiation site has been solved at 2.3 A resolution in two different space groups. It reveals a 22 base pair extended duplex with two noncanonical Watson-Crick-like G-A mismatches, each adjacent to a bulged-out adenine. The structure shows significant asymmetry in deep groove width and G-A base-pair conformations. A network of eight magnesium cations was clearly identified, one being unusually chelated by the 3' phosphate of each bulge across an extremely narrowed deep major groove. CONCLUSIONS: These crystal structures represent the putative matured form of the initial kissing-loop complex. They show the ability of this self-complementary RNA hairpin loop to acquire a more stable extended duplex structure. Both bulged adenines form a striking 'base grip' that could be a recognition signal, either in cis for another viral RNA sequence, or in trans for a protein, possibly the NCp. Magnesium binding might be important to promote and stabilize the observed extrahelical conformation of these bulges.     

Crystal structure of a bulged RNA tetraplex at 1.1 a resolution: implications for a novel binding site in RNA tetraplex

Bulges are an important structural motif in RNA and can be used as recognition and interaction sites in RNA-protein interaction and RNA-RNA interaction. Here we report the first crystal structure of a bulged RNA tetraplex at 1.1 A resolution. The hexamer r(U)(BrdG)r(UGGU) forms a parallel tetraplex with the uridine sandwiched by guanines bulging out. The bulged uridine adopts the syn glycosidic conformation and its O2 and N3 atoms face outwards, serving as an effective recognition and interaction site. The bulge formation both widens the groove width and changes the groove hydrogen-bonding pattern on its 5' side. However, the bulge does not make any bends or kinks in the tetraplex structure. The present study demonstrates the dramatic difference between uridine and guanine in forming tetraplex structure. In addition, both G(syn) tetrad and G(anti) tetrad have been observed. They display the same base-pairing pattern and similar C1'-C1' distance but different hydrogen-bonding patterns in the groove.     

Solution structure of the SL1 RNA of the M1 double-stranded RNA virus of Saccharomyces cerevisiae

The 20-nucleotide SL1 VBS RNA, 5'-GGAGACGC[GAUUC]GCGCUCC (bulged A underlined and loop bases in brackets), plays a crucial role in viral particle binding to the plus strand and packaging of the RNA. Its structure was determined by NMR spectroscopy. Structure calculations gave a precisely defined structure, with an average pairwise root mean square deviation (RMSD) of 1.28 A for the entire molecule, 0.57 A for the loop region (C8-G14), and 0.46 A for the bulge region (G4-G7, C15-C17). Base stacking continues for three nucleotides on the 5' side of the loop. The final structure contains a single hydrogen bond involving the guanine imino proton and the carbonyl O(2) of the cytosine between the nucleotides on the 5' and 3' ends of the loop, although they do not form a Watson-Crick base pair. All three pyrimidine bases in the loop point toward the major groove, which implies that Cap-Pol protein may recognize the major groove of the SL1 loop region. The bulged A5 residue is stacked in the stem, but nuclear Overhauser enhancements (NOEs) suggest that A5 spends part of the time in the bulged-out conformation. The rigid conformation of the upper stem and loop regions may allow the SL1 VBS RNA to interact with Cap-Pol protein without drastically changing its own conformation.     

Crystal structure of paromomycin docked into the eubacterial ribosomal decoding A site

BACKGROUND: Aminoglycoside antibiotics interfere with translation in both gram-positive and gram-negative bacteria by binding to the tRNA decoding A site of the 16S ribosomal RNA. RESULTS: Crystals of complexes between oligoribonucleotides incorporating the sequence of the ribosomal A site of Escherichia coli and the aminoglycoside paromomycin have been solved at 2.5 A resolution. Each RNA fragment contains two A sites inserted between Watson-Crick pairs. The paromomycin molecules interact in an enlarged deep groove created by two bulging and one unpaired adenines. In both sites, hydroxyl and ammonium side chains of the antibiotic form 13 direct hydrogen bonds to bases and backbone atoms of the A site. In the best-defined site, 8 water molecules mediate 12 other hydrogen bonds between the RNA and the antibiotics. Ring I of paromomycin stacks over base G1491 and forms pseudo-Watson-Crick contacts with A1408. Both the hydroxyl group and one ammonium group of ring II form direct and water-mediated hydrogen bonds to the U1495oU1406 pair. The bulging conformation of the two adenines A1492 and A1493 is stabilized by hydrogen bonds between phosphate oxygens and atoms of rings I and II. The hydrophilic sites of the bulging A1492 and A1493 contact the shallow groove of G=C pairs in a symmetrical complex. CONCLUSIONS: Water molecules participate in the binding specificity by exploiting the antibiotic hydration shell and the typical RNA water hydration patterns. The observed contacts rationalize the protection, mutation, and resistance data. The crystal packing mimics the intermolecular contacts induced by aminoglycoside binding in the ribosome.     

Crystal structure of an adenine bulge in the RNA chain of a DNA.RNA hybrid, d(CTCCTCTTC).r(gaagagagag)

Crystal structure of a DNA.RNA hybrid, d(CTCCTCTTC).r(gaagagagag), with an adenine bulge in the polypurine RNA strand was determined at 2.3 A resolution. The structure was solved by the molecular replacement method and refined to a final R-factor of 19.9% (Rfree 22.2%). The hybrid duplex crystallized in the space group I222 with unit cell dimensions, a = 46.66 A, b = 47.61 A and c = 54.05 A, and adopts the A-form conformation. All RNA and DNA sugars are in the C3'-endo conformation, the glycosyl angles in anti conformation and the majority of the C4'-C5' torsion angles in g+ except two trans angles, in conformity with the C3'-endo rigid nucleotide hypothesis. The adenine bulge is looped out and it is also in the anti C3'-endo conformation. The bulge is involved in a base-triple (C.g)*a interaction with the end base-pair (C9.g10) in the minor groove of a symmetry-related molecule. The 2' hydroxyl group of g15 is hydrogen bonded to O2P and O5' of g17, skipping the bulged adenine a16 and stabilizing the sugar-phosphate backbone of the hybrid. The hydrogen bonding and the backbone conformation at the bulged adenine site is very similar to that found in the crystal structure of a protein-RNA complex.     

RNA recognition by Tat-derived peptides: interaction in the major groove?

Replication of human immunodeficiency virus requires binding of the viral Tat protein to its RNA target sequence TAR; peptides derived from Tat bind to a TAR "contact site" spanning 5 bp and a trinucleotide pyrimidine bulge. We find that high affinity binding requires a U residue in the bulge loop and 2 specific adjacent base pairs. Other bulged RNAs bind in a lower affinity nonspecific manner; sequence-specific binding requires a bulge loop of more than 1 nucleotide. Reaction with diethyl pyrocarbonate indicates that one effect of the bulge is to make the otherwise deep and narrow RNA major groove accessible. A model consistent with these data involves local distortion of A-form geometry at the bulge, which bends the helix and permits protein binding and interactive access in the RNA major groove.     

Evolutionary conserved nucleotides within the E.coli 4.5S RNA are required for association with P48 in vitro and for optimal function in vivo.

E.coli 4.5S RNA is homologous to domain IV of eukaryotic SPR7S RNA, the RNA component of the signal recognition particle. The 4.5S RNA is associated in vivo with a 48kD protein (P48), which is homologous to a protein component of the signal recognition particle, SRP54. In addition to secondary structural features, a number of nucleotides are conserved between the 4.5S RNA and domain IV of all other characterised SRP-like RNAs from eubacteria, arachaebacteria and eukaryotes. This domain consists of an extended stem-loop structure; conserved nucleotides lie within the terminal loop and within single-stranded regions bulged from the stem immediately preceding the loop. This conserved region is a candidate for the SRP54/P48 binding site. To determine the functional importance of this region within the 4.5S RNA, mutations were introduced into the 4.5S RNA coding sequence. Mutated alleles were tested for their function in vivo and for the ability of the corresponding RNAs to bind P48 in vitro. Single point mutations in conserved nucleotides within the terminal tetranucleotide loop do not affect P48 binding in vitro and produce only slight growth defects. This suggests that the sequence of the loop may be important for the structure of the molecule rather than for specific interactions with P48. On the other hand, nucleotides within the single-stranded regions bulged from the stem were found to be important both for the binding of P48 to the RNA and for optimal function of the RNA in vivo.     

Solution structure of the pseudo-5' splice site of a retroviral splicing suppressor

Control of Rous sarcoma virus RNA splicing depends in part on the interaction of U1 and U11 snRNPs with an intronic RNA element called the negative regulator of splicing (NRS). A 23mer RNA hairpin (NRS23) of the NRS directly binds U1 and U11 snRNPs. Mutations that disrupt base-pairing between the loop of NRS23 and U1 snRNA abolish its negative control of splicing. We have determined the solution structure of NRS23 using NOEs, torsion angles, and residual dipolar couplings that were extracted from multidimensional heteronuclear NMR spectra. Our structure showed that the 6-bp stem of NRS23 adopts a nearly A-form duplex conformation. The loop, which consists of 11 residues according to secondary structure probing, was in a closed conformation. U913, the first residue in the loop, was bulged out or dynamic, and loop residues G914-C923, G915-U922, and U916-A921 were base-paired. The remaining UUGU tetraloop sequence did not adopt a stable structure and appears flexible in solution. This tetraloop differs from the well-known classes of tetraloops (GNRA, CUYG, UNCG) in terms of its stability, structure, and function. Deletion of the bulged U913, which is not complementary to U1 snRNA, increased the melting temperature of the RNA hairpin. This hyperstable hairpin exhibited a significant decrease in binding to U1 snRNP. Thus, the structure of the NRS RNA, as well as its sequence, is important for interaction with U1 snRNP and for splicing suppression.     

The NMR structure of the 5S rRNA E-domain-protein L25 complex shows preformed and induced recognition

The structure of the complex between ribosomal protein L25 and a 37 nucleotide RNA molecule, which contains the E-loop and helix IV regions of the E-domain of Escherichia coli 5S rRNA, has been determined to an overall r.m.s. displacement of 1.08 A (backbone heavy atoms) by heteronuclear NMR spectroscopy (Protein Databank code 1d6k). The interacting molecular surfaces are bipartite for both the RNA and the protein. One side of the six-stranded beta-barrel of L25 recognizes the minor groove of the E-loop with very little change in the conformations of either the protein or the RNA and with the RNA-protein interactions occurring mainly along one strand of the E-loop duplex. This minor groove recognition module includes two parallel beta-strands of L25, a hitherto unknown RNA binding topology. Binding of the RNA also induces conversion of a flexible loop to an alpha-helix in L25, the N-terminal tip of which interacts with the widened major groove at the E-loop/helix IV junction of the RNA. The structure of the complex reveals that the E-domain RNA serves as a preformed docking partner, while the L25 protein has one preformed and one induced recognition module.     

A new NMR solution structure of the SL1 HIV-1Lai loop-loop dimer

Dimerization of genomic RNA is directly related with the event of encapsidation and maturation of the virion. The initiating sequence of the dimerization is a short autocomplementary region in the hairpin loop SL1. We describe here a new solution structure of the RNA dimerization initiation site (DIS) of HIV-1_Lai. NMR pulsed field-gradient spin-echo techniques and multidimensional heteronuclear NMR spectroscopy indicate that this structure is formed by two hairpins linked by six Watson–Crick GC base pairs. Hinges between the stems and the loops are stabilized by intra and intermolecular interactions involving the A8, A9 and A16 adenines. The coaxial alignment of the three A-type helices present in the structure is supported by previous crystallography analysis but the A8 and A9 adenines are found in a bulged in position. These data suggest the existence of an equilibrium between bulged in and bulged out conformations in solution.     

Fluorescence modification of Escherichia coli 5S RNA

Reaction of 5S RNA with chlorocetaldehyde leads to the conversion of unpaired adenines to the fluorescent 1,N6-etheno-adenine derivatives. Up to 16 of the 23 adenines in free 5S RNA can be modified, the fastest reacting are A29, A34, A57-59. Partial modification of adenines in this area results in a 20% reduction in the efficiency of 5S RNA incorporation into 50S subunits during reconstitution and a 15% reduction in the activity of these subunits in peptide synthesis. Fluorescence from 1,N6-etheno-adenine is quenched in free 5S RNA and is not detectably further influenced by the binding of proteins E-L5, E-L18 and E-L25, nor by the first stage of the two step E. coli 50S subunit reconstitution procedure. However, the fluorescence is further reduced to near zero after the second step of the reconstitution. Thus, 5S RNS free in solution contains 16 unpaired adenines, those in the region between A29 and A59 particularly accessible to modification by chlorocetaldehyde. This portion of the 5S RNA molecule appears to undergo either a conformational change or interacts with other ribosomal components in the last stage of subunit reassembly.     

Characterization of the solution conformations of unbound and Tat peptide-bound forms of HIV-1 TAR RNA.

Basic peptides from the carboxy terminus of the HIV-1 Tat protein bind to the apical stem-loop region of TAR RNA with high affinity and moderate specificity. The conformations of the unbound and 24 residue Tat peptide (Tfr24)-bound forms of TAR RNA have been characterized by NMR spectroscopy. The unbound form of TAR exists in major and minor forms having different trinucleotide bulge conformations. A specific TAR RNA conformational change is observed upon complex formation with Tfr24, consisting of coaxial stacking of helical stems and base triple formation. A U23-A27-U38 base triple is proposed based on exchangeable proton NMR data, where U23 forms a base pair with A27 in the major groove. No evidence for base triple formation was found for Tat peptides in which lysine residues are extensively substituted for arginine.     

Solution structure of the apical stem-loop of the human hepatitis B virus encapsidation signal

Hepatitis B virus (HBV) replication is initiated by HBV RT binding to the highly conserved encapsidation signal, epsilon, at the 5′ end of the RNA pregenome. Epsilon contains an apical stem–loop, whose residues are either totally conserved or show rare non-disruptive mutations. Here we present the structure of the apical stem–loop based on NOE, RDC and ¹H chemical shift NMR data. The ¹H chemical shifts proved to be crucial to define the loop conformation. The loop sequence 5′-CUGUGC-3′ folds into a UGU triloop with a CG closing base pair and a bulged out C and hence forms a pseudo-triloop, a proposed protein recognition motif. In the UGU loop conformations most consistent with experimental data, the guanine nucleobase is located on the minor groove face and the two uracil bases on the major groove face. The underlying helix is disrupted by a conserved non-paired U bulge. This U bulge adopts multiple conformations, with the nucleobase being located either in the major groove or partially intercalated in the helix from the minor groove side, and bends the helical stem. The pseudo-triloop motif, together with the U bulge, may represent important anchor points for the initial recognition of epsilon by the viral RT.     

Structure analysis of free and bound states of an RNA aptamer against ribosomal protein S8 from Bacillus anthracis

Several protein-targeted RNA aptamers have been identified for a variety of applications and although the affinities of numerous protein-aptamer complexes have been determined, the structural details of these complexes have not been widely explored. We examined the structural accommodation of an RNA aptamer that binds bacterial r-protein S8. The core of the primary binding site for S8 on helix 21 of 16S rRNA contains a pair of conserved base triples that mold the sugar-phosphate backbone to S8. The aptamer, which does not contain the conserved sequence motif, is specific for the rRNA binding site of S8. The protein-free RNA aptamer adopts a helical structure with multiple non-canonical base pairs. Surprisingly, binding of S8 leads to a dramatic change in the RNA conformation that restores the signature S8 recognition fold through a novel combination of nucleobase interactions. Nucleotides within the non-canonical core rearrange to create a G-(G-C) triple and a U-(A-U)-U quartet. Although native-like S8-RNA interactions are present in the aptamer-S8 complex, the topology of the aptamer RNA differs from that of the helix 21-S8 complex. This is the first example of an RNA aptamer that adopts substantially different secondary structures in the free and protein-bound states and highlights the remarkable plasticity of RNA secondary structure.