Protein-rRNA binding features and their structural and functional implications in ribosomes as determined by cross-linking studies

We have investigated protein-rRNA cross-links formed in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus at the molecular level using UV and 2-iminothiolane as cross-linking agents. We identified amino acids cross-linked to rRNA for 13 ribosomal proteins from these organisms, namely derived from S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29 and L36. Several other peptide stretches cross-linked to rRNA have been sequenced in which no direct cross-linked amino acid could be detected. The cross-linked amino acids are positioned within loop domains carrying RNA binding features such as conserved basic and aromatic residues. One of the cross-linked peptides in ribosomal protein S3 shows a common primary sequence motif--the KH motif--directly involved in interaction with rRNA, and the cross-linked amino acid in ribosomal protein L36 lies within the zinc finger-like motif of this protein. The cross-linked amino acids in ribosomal proteins S17 and L6 prove the proposed RNA interacting site derived from three-dimensional models. A comparison of our structural data with mutations in ribosomal proteins that lead to antibiotic resistance, and with those from protein-antibiotic cross-linking experiments, reveals functional implications for ribosomal proteins that interact with rRNA.     

Unique wave front for dendritic spines with Nagumo dynamics

Expression of the Gag-Pol polyprotein of Rous sarcoma virus (RSV) requires a -1 ribosomal frameshifting event at the overlap region of the gag and pol open reading frames. The signal for frameshifting is composed of two essential mRNA elements; a slippery sequence (AAAUUUA) where the ribosome changes reading frame, and a stimulatory RNA structure located immediately downstream. This RNA is predicted to be a complex stem-loop but may also form an RNA pseudoknot. We have investigated the structure of the RSV frameshift signal by a combination of enzymatic and chemical structure probing and site-directed mutagenesis. The stimulatory RNA is indeed a complex stem-loop with a long stable stem and two additional stem-loops contained as substructures within the main loop region. The substructures are not however required for frameshifting. Evidence for an additional interaction between a stretch of nucleotides in the main loop and a region downstream to generate an RNA pseudoknot was obtained from an analysis of the frameshifting properties of RSV mutants translated in the rabbit reticulocyte lysate in vitro translation system. Mutations that disrupted the predicted pseudoknot-forming sequences reduced frameshifting but when the mutations were combined and should re-form the pseudoknot, frameshifting was restored to a level approaching that of the wild-type construct. It was also observed that the predicted pseudoknot-forming regions had reduced sensitivity to cleavage by the single-stranded probe imidazole. Overall, however, the structure probing data indicate that the pseudoknot interaction is weak and may form transiently. In comparison to other characterised RNA structures present at viral frameshift signals, the RSV stimulator falls into a novel group. It cannot be considered to be a simple hairpin-loop yet it is distinct from other well characterised frameshift-inducing RNA pseudoknots in that the overall contribution of the RSV pseudoknot to frameshifting is less dramatic.     

Effects of the novel neuroprotective agent, riluzole, on human brain function and behavior: I. Double-blind, placebo-controlled EEG mapping and psychometric studies under normoxia

BACKGROUND: Detailed structural information on ribosomal proteins has increased our understanding of the structure, function and evolution of the ribosome. L14 is one of the most conserved ribosomal proteins and appears to have a central role in the ribonucleoprotein complex. Studies have indicated that L14 occupies a central location between the peptidyl transferase and GTPase regions of the large ribosomal subunit. RESULTS: The crystal structure of L14 from Bacillus stearothermophilus has been solved using a combination of isomorphous replacement and multiwavelength anomalous dispersion (MAD) methods. The structure comprises a five-stranded beta-barrel, a C-terminal loop region that contains two small alpha-helices, and a beta-ribbon that projects from the beta-barrel. An analysis of the structure and the conserved amino acids reveals three surface patches that probably mediate L14-RNA and L14-protein interactions within the ribosome. CONCLUSIONS: The accepted role of ribosomal proteins is to promote the folding and stabilization of ribosomal RNA. The L14 structure is consistent with this notion, and it suggests that the RNA binds in two sites. One RNA-binding site appears to recognize a distinct region of ribosomal RNA during particle assembly. The second site is smaller and may become occupied during the later compaction of the RNA. The surface hydrophobic patch is a likely site of protein-protein interaction, possibly with L19.     

The ribosomal environment of tRNA: crosslinks to rRNA from positions 8 and 20:1 in the central fold of tRNA located at the A, P, or E site

The naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl) uridine ("acp3U") at position 20:1 of lupin tRNAMet was coupled to a photoreactive diazirine derivative. Similarly, the 4-thiouridine at position 8 of Escherichia coli tRNAPhe was modified with an aromatic azide. Each of the derivatized tRNAs was bound to E. coli ribosomes in the presence of suitable mRNA analogues, under conditions specific for the A, P, or E sites. After photoactivation of the diazirine or azide groups, the sites of crosslinking from the tRNAs to 16S or 23S rRNA were analyzed by our standard procedures, involving a combination of ribonuclease H digestion and primer extension analysis. The crosslinked ribosomal proteins were also identified. The results for the rRNA showed a well-defined series of crosslinks to both the 16S and 23S molecules, the most pronounced being (1) an entirely A-site-specific crosslink from tRNA position 20:1 to the loop-end region (nt 877-913) of helix 38 of the 23S RNA (a region that has not so far been associated at all with tRNA binding), and (2) a largely P-site-specific crosslink from tRNA position 8 to nt 2111-2112 of the 23S RNA (nt 2112 being a position that has previously been identified in footprinting studies as belonging to the ribosomal E site). The data are compared with results from a parallel study of crosslinks from position 47 (also in the central fold of the tRNA), as well as with previously published crosslinks from the anticodon loop (positions 32, 34, and 37) and the CCA-end region (position 76, and the aminoacyl residue).     

Depurination of A4256 in 28 S rRNA by the ribosome-inactivating proteins from barley and ricin results in different ribosome conformations

Ribosomal function in protein synthesis requires dynamic flexibility of the ribosomal structure. The two translational inhibitors derived from seeds of ricin and barley destroy the dynamic properties of the ribosome by selective depurination of A4256 in the phylogenetically conserved alpha-sarcin/ricin loop of mouse 28 S rRNA. As the alpha-sarcin/ricin loop is involved in binding of elongation factors to the ribosome, depurination blocks the protein synthesis elongation cycle. Depurination by the barley translational inhibitor (BTI) mainly effects eEF-1 alpha related functions, while ricin interferes with the interaction of eEF-2 with the ribosome. Analysis of the ribosomal structure after inhibitor shows that the accessibility of the rRNAs for single-strand-specific chemical modification was altered. Reactivity changes were seen in domains I, II and V of 28 S rRNA and in 5 S rRNA. A majority of the reactivity changes were found in putative functional regions of the rRNAs, such as the regions involved in peptidyltransferase activity, subunit interaction and in the binding of elongation factors. Most of the observed structural changes made the rRNAs less accessible for chemical modification, suggesting that the ribosomal particles became less flexible after inhibitor treatment. Moreover, the modification patterns obtained from the two inhibitor-treated ribosomal particles were only partly overlapping, indicating that the structure of the large ribosomal subunit differed after ricin and BTI treatment. Surprisingly, depurination in the alpha-sarcin/ricin loop of 28 S rRNA also affected the structure of the 3' major domain in 18 S rRNA.     

The 5S rRNA loop E: chemical probing and phylogenetic data versus crystal structure

A significant fraction of the bases in a folded, structured RNA molecule participate in noncanonical base pairing interactions, often in the context of internal loops or multi-helix junction loops. The appearance of each new high-resolution RNA structure provides welcome data to guide efforts to understand and predict RNA 3D structure, especially when the RNA in question is a functionally conserved molecule. The recent publication of the crystal structure of the "Loop E" region of bacterial 5S ribosomal RNA is such an event [Correll CC, Freeborn B, Moore PB, Steitz TA, 1997, Cell 91:705-712]. In addition to providing more examples of already established noncanonical base pairs, such as purine-purine sheared pairings, trans-Hoogsteen UA, and GU wobble pairs, the structure provides the first high-resolution views of two new purine-purine pairings and a new GU pairing. The goal of the present analysis is to expand the capabilities of both chemical probing and phylogenetic analysis to predict with greater accuracy the structures of RNA molecules. First, in light of existing chemical probing data, we investigate what lessons could be learned regarding the interpretation of this widely used method of RNA structure probing. Then we analyze the 3D structure with reference to molecular phylogeny data (assuming conservation of function) to discover what alternative base pairings are geometrically compatible with the structure. The comparisons between previous modeling efforts and crystal structures show that the intricate involvements of ions and water molecules in the maintenance of non-Watson-Crick pairs render the process of correctly identifying the interacting sites in such pairs treacherous, except in cases of trans-Hoogsteen A/U or sheared A/G pairs for the adenine N1 site. The phylogenetic analysis identifies A/A, A/C, A/U and C/A, C/C, and C/U pairings isosteric with sheared A/G, as well as A/A and A/C pairings isosteric with both G/U and G/G bifurcated pairings. Thus, each non-Watson-Crick pair could be characterized by a phylogenetic signature of variations between isosteric-like pairings. In addition to the conservative changes, which form a dictionary of pairings isosterically compatible with those observed in the crystal structure, concerted changes involving several base pairs also occur. The latter covariations may indicate transitions between related but distinctive motifs within the loop E of 5S ribosomal RNA.     

Peptidyl-transferase ribozymes: trans reactions, structural characterization and ribosomal RNA-like features

BACKGROUND: One of the most significant questions in understanding the origin of life concerns the order of appearance of DNA, RNA and protein during early biological evolution. If an 'RNA world' was a precursor to extant life, RNA must be able not only to catalyze RNA replication but also to direct peptide synthesis. Iterative RNA selection previously identified catalytic RNAs (ribozymes) that form amide bonds between RNA and an amino acid or between two amino acids. RESULTS: We characterized peptidyl-transferase reactions catalyzed by two different families of ribozymes that use substrates that mimic A site and P site tRNAs. The family II ribozyme secondary structure was modeled using chemical modification, enzymatic digestion and mutational analysis. Two regions resemble the peptidyl-transferase region of 23S ribosomal RNA in sequence and structural context; these regions are important for peptide-bond formation. A shortened form of this ribozyme was engineered to catalyze intermolecular ('trans') peptide-bond formation, with the two amino-acid substrates binding through an attached AMP or oligonucleotide moiety. CONCLUSIONS: An in vitro-selected ribozyme can catalyze the same type of peptide-bond formation as a ribosome; the ribozyme resembles the ribosome because a very specific RNA structure is required for substrate binding and catalysis, and both amino acids are attached to nucleotides. It is intriguing that, although there are many different possible peptidyl-transferase ribozymes, the sequence and secondary structure of one is strikingly similar to the 'helical wheel' portion of 23S rRNA implicated in ribosomal peptidyl-transferase activity.     

Structural studies of ribosomal proteins

Crystal and solution structures of fourteen ribosomal proteins from thermophilic bacteria have been determined during the last decade. This paper reviews structural studies of ribosomal proteins from Thermus thermophilus carried out at the Institute of Protein Research (Pushchino, Russia) in collaboration with the University of Lund (Lund, Sweden) and the Center of Structural Biochemistry (Karolinska Institute, Huddinge, Sweden). New experimental data on the crystal structure of the ribosomal protein L30 from T. thermophilus are also included.     

Involvement of the Xenopus laevis Ro60 autoantigen in the alternative interaction of La and CNBP proteins with the 5'UTR of L4 ribosomal protein mRNA

In vertebrates the synthesis of ribosomal proteins is co-ordinately regulated at the translational level. The 5'-untranslated region (5'UTR) of this class of mRNAs contains conserved regions that are necessary and sufficient for translational regulation. Recently, we found that two proteins, the Xenopus laevis La autoantigen and the cellular nucleic acid binding protein (CNBP), are able to bind in vitro a pyrimidine tract at the 5' end and a downstream region, respectively. These regions are considered the common cis-acting elements of translational regulation. It was previously observed that the binding of both these putative trans-acting factors to their RNA sequences is assisted by a protease-sensitive factor(s) that dissociates from the complex after its formation. Here we provide evidence that the requirement for an ancillary factor assisting La binding to the pyrimidine tract of ribosomal protein mRNAs is typical of this RNA, and secondly that it may involve an RNA recognition motif of the La protein not clearly characterized previously. We also show that the Ro60 autoantigen is involved in the common factor activity necessary for the binding of La and CNBP proteins to their respective sequences. In addition, our findings suggest that an RNA also participates in this process. We show that CNBP can multimerise and that it binds to the 5'UTR as a dimer. Both La and CNBP compete for the interaction with the factor, and their binding to the 5'UTR is mutually exclusive. Our results from the binding analysis of mutations in the 5'UTR, which are known to disrupt the translational control in vivo, suggest a model in which the protein interactions and the 5'UTR RNA structure may co-operate in regulating the translational fate of ribosomal protein mRNAs.     

Central sleep apnoea and heart failure (part II)

The three-dimensional (3-D) structure of a RNA pseudoknot that causes the efficient ribosomal frameshifting in the gag-pro region of mouse mammary tumor virus (MMTV) has been determined recently by nuclear magnetic resonance (NMR) studies. But since the structure refinement in the studies did not use metal ions and waters, it is not clear how metal ions participate in the stabilization of the pseudoknot, and what kind of ion-RNA interactions dominate in the tertiary contacts for the RNA pseudoknotting. Based on the reported structure data of the pseudoknot VPK of MMTV, we gradually refined the structure by restrained molecular dynamics (MD) using NMR distance restraints. Restrained MD simulation of the RNA pseudoknot was performed with sodium ions and water molecules. Our results are in good agreement with known NMR data and delineate the importance of the metal ion coordination in the stability of the pseudoknot. In the non-coaxially stacking pseudoknot, stem 1 (S1), stem 2 (S2), and the intervening A14 involves unconventional stacking of base pairs coordinated by Na+ and/or bridging water molecules. A6 and G7 of loop L1 make a perfect base stacking in the major groove and are further stabilized by coordinated Na+ ions and water molecules. The first 4-nucleotide (nt) ACUC of loop L2 form a sharp turn and the following 4-nt AAAA cross the minor groove of S1 and are steadied by interactions with the nucleotides of S , bridging water molecules and coordinated Na+ ions. Our studies suggest that the metal ion plays a crucial role in the RNA pseudoknotting of VPK. In the stacking interior of S1 and S2, the Na+ ion is positioned in the major groove and interacts directly with the carbonyl group O6 of G28 and carbonyl group O4 of U13 in the wobble base pair U13:G28. The ion-RNA interactions in MMTV VPK not only stabilize the RNA pseudoknot but also modify the electrostatic properties of the nucleotides at the critical parts of the pseudoknot VPK.     

RNA sequence determinants for aminoglycoside binding to an A-site rRNA model oligonucleotide

The codon-anticodon interaction on the ribosome occurs in the A site of the 30 S subunit. Aminoglycoside antibiotics, which bind to ribosomal RNA in the A site, cause misreading of the genetic code and inhibit translocation. Biochemical studies and nuclear magnetic resonance spectroscopy were used to characterize the interaction between the aminoglycoside antibiotic paromomycin and a small model oligonucleotide that mimics the A site of Escherichia coli 16 S ribosomal RNA. Upon chemical modification, the RNA oligonucleotide exhibits an accessibility pattern similar to that of 16 S rRNA in the 30 S subunit. In addition, the oligonucleotide binds specifically aminoglycoside antibiotics. The antibiotic binding site forms an asymmetric internal loop, caused by non-canonical base-pairs. Nucleotides that are important for binding of paromomycin were identified by performing quantitative footprinting on oligonucleotide sequence variants and include the C1407.G1494 base-pair, and A.U base-pair at positions 1410/1490, and nucleotides A1408, A1493 and U1495. The asymmetry of the internal loop, which requires the presence of a nucleotide in position 1492, is also crucial for antibiotic binding. Introduction into the oligonucleotide of base changes that are known to confer aminoglycoside resistance in 16 S rRNA result in weaker binding of paromomycin to the oligonucleotide. Oligonucleotides homologous to eukaryotic rRNA sequences show reduced binding of paromomycin, suggesting a physical origin for the species-specific action of aminoglycosides.     

Downregulated expression of the signaling molecules Nck, c-Crk, Grb2/Ash, PI 3-kinase p110 alpha and WRN during fibroblast aging in vitro

The synthetic RNA fragment 5'-CUGGGCGG(GCGA)CCGCCUGG (nucleotides in parentheses indicate the loop region) corresponds to the natural sequence of domain E from nucleotides 79-97 of the Thermus flavus 5S rRNA including a hairpin loop. The RNA structure determined at 3.0 A and refined to an R-value of 24.1% also represents the first X-ray structure GNRA tetraloop. The loop is in distinctly different conformation from other GNRA tetraloops analyzed by NMR. The conformation of the two molecules in the asymmetric unit is influenced and stabilized by specific intermolecular contacts. The structural features presented here give evidence for the ability of RNA molecules to adapt to specific environments.     

The crystal structure of ribosomal protein L22 from Thermus thermophilus: insights into the mechanism of erythromycin resistance

BACKGROUND:. The ribosomal protein L22 is one of five proteins necessary for the formation of an early folding intermediate of the 23S rRNA. L22 has been found on the cytoplasmic side of the 50S ribosomal subunit. It can also be labeled by an erythromycin derivative bound close to the peptidyl-transfer center at the interface side of the 50S subunit, and the amino acid sequence of an erythromycin-resistant mutant is known. Knowing the structure of the protein may resolve this apparent conflict regarding the location of L22 on the ribosome. RESULTS:. The structure of Thermus thermophilus L22 was solved using X-ray crystallography. L22 consists of a small alpha+beta domain and a protruding beta hairpin that is 30 A long. A large part of the surface area of the protein has the potential to be involved in interactions with rRNA. A structural similarity to other RNA-binding proteins is found, possibly indicating a common evolutionary origin. CONCLUSIONS:. The extensive surface area of L22 has the characteristics of an RNA-binding protein, consistent with its role in the folding of the 23S rRNA. The erythromycin-resistance conferring mutation is located in the protruding beta hairpin that is postulated to be important in L22-rRNA interactions. This region of the protein might be at the erythromycin-binding site close to the peptidyl transferase center, whereas the opposite end may be exposed to the cytoplasm.     

Metal ion probing of rRNAs: evidence for evolutionarily conserved divalent cation binding pockets

Ribosomes are multifunctional RNP complexes whose catalytic activities absolutely depend on divalent metal ions. It is assumed that structurally and functionally important metal ions are coordinated to highly ordered RNA structures that form metal ion binding pockets. One potent tool to identify the structural surroundings of high-affinity metal ion binding pockets is metal ion-induced cleavage of RNA. Exposure of ribosomes to divalent metal ions, such as Pb2+, Mg2+, Mn2+, and Ca2+, resulted in site-specific cleavage of rRNAs. Sites of strand scission catalyzed by different cations accumulate at distinct positions, indicating the existence of general metal ion binding centers in the highly folded rRNAs in close proximity to the cleavage sites. Two of the most efficient cleavage sites are located in the 5' domain of both 23S and 16S rRNA, regions that are known to self-fold even in the absence of ribosomal proteins. Some of the efficient cleavage sites were mapped to the peptidyl transferase center located in the large ribosomal subunit. Furthermore, one of these cleavages was clearly diminished upon AcPhe-tRNA binding to the P site, but was not affected by uncharged tRNA. This provides evidence for a close physical proximity of a metal ion to the amino acid moiety of charged tRNAs. Interestingly, comparison of the metal ion cleavage pattern of eubacterial 70S with that of human 80S ribosomes showed that certain cleavage sites are evolutionarily highly conserved, thus demonstrating an identical location of a nearby metal ion. This suggests that cations, bound to evolutionarily constrained binding sites, are reasonable candidates for being of structural or functional importance.     

Stability of the TAR RNA and its complex with arginine

Most stable secondary structures and their stabilization energies of the TAR RNA with +1 to +104 nucleotide-sequence region were calculated at different temperatures by using thermodynamic parameters for RNA structure prediction. The most stable secondary structure has one bulge and one loop within the region of +20 to +40 nucleotide sequence, and its stabilization energy at 37 degrees C was -46.3 kcal mol-1. The interaction of a TAR bulge oligomer (TARBO) with arginine (Arg) which was in the binding site of a Tat protein was also investigated by CD measurements. The addition of Arg did not affect the CD spectrum of TARBO. The result was different from that of the RNA oligomer with both bulge and loop.     

In vitro selection of dopamine RNA ligands

RNA aptamers that specifically bind dopamine have been isolated by in vitro selection from a pool of 3.4 x 10(14) different RNA molecules. One aptamer (dopa2), which dominated the selected pool, has been characterized and binds to the dopamine affinity column with a dissociation constant of 2.8 microM. The specificity of binding has been determined by studying binding properties of a number of dopamine-related molecules, showing that the interaction with the RNA might be mediated by the hydroxyl group at position 3 and the proximal aliphatic chain in the dopamine molecule. The binding domain was initially localized by boundary experiments. Further definition of the dopamine binding site was obtained by secondary selection on a pool of sequences derived from a partial randomization of the dopa2 molecule. Sequence comparison of a large panel of selected variants revealed a structural consensus motif among the active aptamers. The dopamine binding pocket is built up by a tertiary interaction between two stem and loop motifs, creating a stable framework in which five invariant nucleotides are precisely arrayed. Minimal active sequence and key nucleotides have been confirmed by the design of small functional aptamers and mutational analysis. Enzymatic probing suggests that the RNA might undergo a conformational change upon ligand binding that stabilizes the proposed tertiary structure.     

Systematic, computer-assisted optimisation of the isolation of Thermus thermophilus 50S ribosomal proteins by reversed-phase high-performance liquid chromatography

Isolation of the 50S ribosomal proteins from Thermus thermophilus has been achieved for the first time using reversed-phase high-performance liquid chromatography based on the use of the non-end-capped LiChrospher RP-18 sorbent and computer-assisted method development for optimisation of the resolution. The separation approach for these basic ribosomal proteins utilised mobile phases of high ionic strength, to suppress silanophilic interactions with this type of reversed-phase sorbent. These conditions were found to be a key requirement for achieving good resolution with minimal peak-tailing. The retention times of the 50S ribosomal proteins of Thermus thermophilus were observed to be in very close agreement with the values predicted by computer simulation procedures based on linear solvent strength concepts, with an average error of only 0.5%, whilst base-line resolution was achieved for most of the adjacent peak zones. Following N-terminal sequencing, the proteins TthL5, TthL9, TthL18, TthL24, TthL29, TthL32, TthL34, TthL35 and TthL36 of Thermus thermophilus were readily identified. This approach thus provided a readily optimised strategy for the isolation of the 50S ribosomal proteins from Thermus thermophilus and should be generally applicable to the corresponding ribosomal proteins from various other species, as well as other classes of basic proteins present in crude extracts derived from other biological sources.