Minimum sequence requirements for the binding of paromomycin to the rRNA decoding site A

We have recently introduced a computational methodology that combines molecular dynamics (MD) simulations, free-energy calculations, and in vitro binding assays to predict the minimum RNA structural requirements for selective, high-affinity RNA binding to small-molecule ligands. Here, we show that this methodology can be applied to the conformationally flexible aminoglycoside antibiotic paromomycin. A RNA consisting of an 11-mer:10-mer duplex that contains one 16S ribosome RNA decoding A-site bound to paromomycin was simulated for 4 ns. The methodology predicts that the 11-mer:10-mer duplex binds to paromomycin with high affinity, whereas smaller RNA duplexes lose complex stability and the ability to bind paromomycin. The predicted high-affinity binding to paromomycin of the 11-mer:10-mer duplex was confirmed experimentally (EC(50) = 0.28 microM), as well as the inability of smaller complexes to bind. Our simulations show good agreement with experiment for dynamic and structural properties of the isolated A-site, including hydrogen-bonding networks and RNA structural rearrangements upon ligand binding. The results suggest that MD simulations can supplement in vitro methods as a tool for predicting minimum RNA-binding motifs for both small, rigid ligands, and large, flexible ligands when structural information is available.     

Electric birefringence of eukaryotic ribosomes

Eukaryotic 60S ribosomal subunits were studied by transient electric birefringence. Conformations of subunits in active and inactive states upon changes in magnesium concentration were compared by electric birefringence and orientational relaxation time measurements. Active subunits exhibit a positive birefringence and a relaxation time of the order of 8 microseconds. In the presence of EDTA, inactive subunits show no birefringence. When Mg2+ is reverted in the cold to its initial level, the electro-optical properties of the subunits are partially restored although the particles remain biologically inactive.     

G1401: a keystone nucleotide at the decoding site of Escherichia coli 30S ribosomes.

16S ribosomal RNA contains three highly conserved single-stranded regions. Centrally located in one of these regions is the C1400 residue. Zero-length cross-linking of this residue to the anticodon of ribosome-bound tRNA showed that it was at or near the ribosomal decoding site [Ehresmann, C., Ehresmann, B., Millon, R., Ebel, J-P., Nurse, K., & Ofengand, J. (1984) Biochemistry 23, 429-437]. To assess the functional significance of sequence conservation of rRNA in the vicinity of this functionally important site, a series of site-directed mutations in this region were constructed and the effects of these mutations on the partial reactions of protein synthesis determined. Mutation of C1400 or C1402 to any other base only moderately affected a set of in vitro protein synthesis partial reactions. However, any base change from the normal G1401 residue blocked all of the tested ribosomal functions. This was also true for the deletion of G1401. Deletion of C1400 or C1402 had more complex effects. Whereas subunit association was hardly affected, 30S initiation complex formation was blocked by deletion of C1400 but much less so by deletion of C1402. Alternatively, tRNA binding to the ribosomal A site was more strongly affected by deletion of C1402 than by deletion of C1400. P site binding was inhibited by either deletion. HPLC analysis of the in vitro reconstituted mutant ribosomes showed that none of the functional effects were due to the absence or gross reduction in amount of any ribosomal protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Colicin E3 cleavage of 16S rRNA impairs decoding and accelerates tRNA translocation on Escherichia coli ribosomes

The cytotoxin colicin E3 targets the 30S subunit of bacterial ribosomes and specifically cleaves 16S rRNA at the decoding centre, thereby inhibiting translation. Although the cleavage site is well known, it is not clear which step of translation is inhibited. We studied the effects of colicin E3 cleavage on ribosome functions by analysing individual steps of protein synthesis. We find that the cleavage affects predominantly the elongation step. The inhibitory effect of colicin E3 cleavage originates from the accumulation of sequential impaired decoding events, each of which results in low occupancy of the A site and, consequently, decreasing yield of elongating peptide. The accumulation leads to an almost complete halt of translation after reading of a few codons. The cleavage of 16S rRNA does not impair monitoring of codon-anticodon complexes or GTPase activation during elongation-factor Tu-dependent binding of aminoacyl-tRNA, but decreases the stability of the codon-recognition complex and slows down aminoacyl-tRNA accommodation in the A site. The tRNA-mRNA translocation is faster on colicin E3-cleaved than on intact ribosomes and is less sensitive to inhibition by the antibiotic viomycin.     

Immunochemical analysis of the structure of eukaryotic ribosomes

Summary Antibodies were prepared in rabbits and sheep to rat liver ribosomes, ribosomal subunits, and to mixtures of proteins from the particles. The antisera were characterized by quantitative immunoprecipitation, by passive hemagglutination, by immunodiffusion on Ouchterlony plates, and by immunoelectrophoresis. While all the antisera contained antibodies specific for ribosomal proteins, none had precipitating antibodies against ribosomal RNA. Rat liver ribosomal proteins were more immunogenic in sheep than rabbits, and the large ribosomal subunit and its proteins were more immunogenic than those of the 40S subparticle. Antisera specific for one or the other ribosomal subunit could be prepared; thus it is unlikely that there are antigenic determinants common to the proteins of the two subunits. When ribosomes, ribosomal subunits, or mixtures of proteins were used as antigens the sera contained antibodies directed against a large number of the ribosomal proteins.Abbreviations TP total proteins—used to designate mixtures of proteins from ribosomal particles, hence TP80 is a mixtures of all the proteins from 80S ribosomes - TP60 the proteins from 60S subunits - TP40 the proteins from 40S particles     

Structural arrangement of the decoding site of Escherichia coli ribosomes as revealed from the data on affinity labelling of ribosomes by analogs of mRNA--derivatives of oligoribonucleotides

Using derivatives of oligoribonucleotides bearing an active group at the 5'- or 3'-end, the affinity modification of Escherichia coli ribosomes has been investigated in model complexes imitating various steps of initiation and elongation with a different extent of approximation to the real protein-synthesizing system. The protein environment of the ribosome decoding site is determined. The S3, S4, S9, L2, L7/L12 proteins belong to the 5'-region of the decoding site, and the S5, S7, S9, L1, L16 proteins to the 3'-region. In the process of translation the template moves along the external side of the 30 S subunit, from the L1 ridge to the L7/L12 stalk. The structural arrangement of the decoding site or its nearest environment depends on the functional state of ribosomes in the process of translation.     

Identification of a site on 18 S rRNA of human placenta ribosomes in the region of the mRNA binding center

The affinity labeling of human placenta 80 S ribosomes by 4-(N-2-chloroethyl-N-methylamino)benzyl-5'-phosphamide of hexauridylate was studied. This mRNA analog has normal coding properties because its binding to placenta ribosomes significantly increases in the presence of cognate tRNAPhe. Incubation of the mRNA analog in the complex with the ribosomes and Phe-tRNAPhe leads to its covalent attachment exclusively to the small subunit (mainly to 18 S rRNA). The site of the reaction has been identified by hybridization experiments to be located within positions 975 to 1055 of 18 S rRNA. The identified fragment is located in a highly conserved part of the small subunit rRNA domain II.     

Three helical domains form a protein binding site in the 5S RNA-protein complex from eukaryotic ribosomes.

A ribosomal protein binding site in the eukaryotic 5S rRNA has been delineated by examining the effect of sequence variation and nucleotide modification on the RNA's ability to exchange into the EDTA-released, yeast ribosomal 5S RNA-protein complex. 5S RNAs of divergent sequence from a variety of eukaryotic origins could be readily exchanged into the yeast complex but RNA from bacterial origins was rejected. Nucleotide modifications in any of three analogous helical regions in eukaryotic 5S RNAs of differing origin reduced the ability of this RNA molecule to form homologous or heterologous RNA-protein complexes. Because sequence comparisons did not indicate common nucleotide sequences in the interacting helical regions, a model is suggested in which the eukaryotic 5S RNA binding protein does not simply recognize specific nucleotide sequences but interacts with three strategically oriented helical domains or functional groups within these domains. Two of the domains bear a limited sequence homology with each other and contain an unpaired nucleotide or "bulge" similar to that recently reported for one of the 5S RNA binding proteins in Escherichia coli (Peattie, D.A., Douthwaite, S., Garrett, R.A. and Noller, H.F. (1981) Proc. Natl. Acad. Sci. 78, 7331-7335). The results further indicate that the single ribosomal protein of eukaryotic 5S RNA-protein complexes interacts with the same region of the 5S rRNA molecule as do the multiple protein components in complexes of prokaryotic origin.     

Pseudomonas aeruginosa cleaves the decoding center of Caenorhabditis elegans ribosomes

Pathogens such as Pseudomonas aeruginosa advantageously modify animal host physiology, for example, by inhibiting host protein synthesis. Translational inhibition of insects and mammalian hosts by P. aeruginosa utilizes the well-known exotoxin A effector. However, for the infection of Caenorhabditis elegans by P. aeruginosa, the precise pathways and mechanism(s) of translational inhibition are not well understood. We found that upon exposure to P. aeruginosa PA14, C. elegans undergoes a rapid loss of intact ribosomes accompanied by the accumulation of ribosomes cleaved at helix 69 (H69) of the 26S ribosomal RNA (rRNA), a key part of ribosome decoding center. H69 cleavage is elicited by certain virulent P. aeruginosa isolates in a quorum sensing (QS)–dependent manner and independently of exotoxin A–mediated translational repression. H69 cleavage is antagonized by the 3 major host defense pathways defined by the pmk-1, fshr-1, and zip-2 genes. The level of H69 cleavage increases with the bacterial exposure time, and it is predominantly localized in the worm’s intestinal tissue. Genetic and genomic analysis suggests that H69 cleavage leads to the activation of the worm’s zip-2-mediated defense response pathway, consistent with translational inhibition. Taken together, our observations suggest that P. aeruginosa deploys a virulence mechanism to induce ribosome degradation and H69 cleavage of host ribosomes. In this manner, P. aeruginosa would impair host translation and block antibacterial responses.

During infection of the nematode Caenorhabditis elegans by the bacterium Pseudomonas aeruginosa, a bacterial virulence mechanism leads to the cleavage of host ribosomal RNAs at the decoding center, thereby shutting down translation.     

gamma-Carboxyglutamic acid in eukaryotic and prokaryotic ribosomes.

γ-carboxyglutamic acid (GLA) has been assayed in ribosomes of wheat germ and E. coli, and in E. coli ribosomal sub-units, by amino acid analysis of total protein. Results, as nMoles GLA/mg protein, were 18.1 (wheat germ), 58.4 (E. coli), 39.6 and 81.5 (E. coli 30S and 50S sub-units, respectively.) Results for wheat germ and previously reported mammalian ribosomes were highly similar. Hence the level of GLA in eukaryotic ribosomes appears to be approximately constant, but low compared to bacterial (E. coli) ribosomes.     

Defining the eukaryotic cytosolic chaperonin-binding sites in human tubulins

The actins and tubulins are the obligate substrates in vivo of the chaperonin-containing TCP-1 (CCT). The precise elements of recognition between the chaperonin and its substrates remain largely unknown. We have used a solid phase peptide binding assay to screen the human alpha, beta and gamma-tubulin sequences for CCT recognition. Multiple regions seem to be implicated in interactions between tubulins and CCT. These potential CCT-binding sites are highly dispersed throughout the primary sequences of the human tubulins. In addition, using site-directed mutagenesis we assessed the contribution of the selected residues in the C-terminal domain of beta-tubulin to CCT binding. Various hot spots have been identified even though, in each case, their replacement by alanine does not reduce dramatically the total affinity of beta-tubulin for CCT. The CCT-binding information in the tubulins is probably confined to multiple specific regions each having weak or moderate affinity for CCT apical domains. The main binding region seems to be located between residues 263 and 384, but there are no single amino acid residues in this region, which make large contributions to the binding energy, although we have detected a minor contribution by F377. These biochemical results are understandable in the context of our recent structural analysis of CCT-tubulin complexes by cryo-electron microscopy and image reconstruction, which shows that, in one stage of an in vitro binding reaction between apo-CCT and tubulin diluted from guanidinium chloride, ten major, stable contacts between tubulin and CCT are involved. Therefore, specificity is achieved through the co-operation of many specific, albeit weak, interactions.     

Purification of eukaryotic ribosomes by isopycnic centrifugation in sucrose

A simple and inexpensive procedure for the isolation and purification of ribosomes from eukaryotes is described. The method avoids pelleting of ribosomes at high centrifugal forces and involves isopyenic centrifugation of the post-mitochrondrial supernatant in sucrose, precipitation of ribosomes with 10% polyethylene glycol, and zonal sucrose gradient centrifugation. The ribosomes obtained in this way are very pure and thus especially suited for the measurements of physical properties. The isopycnic centrifugation can also be used for the purification of other macromolecules and is only limited by a maximum density of sucrose of 1.40 g/cm³ obtained at the bottom of the centrifugation tubes.     

Biochemical characterization of the ribosomal decoding site

Prior to the emergence of crystal structures of the ribosome, different ribosomal functions were identified with specific regions of ribosomal RNA by biochemical and genetic approaches. In particular, three universally conserved bases of 16S rRNA, G530, A1492 and A1493, were implicated in the interaction of the incoming aminoacyl-tRNA with the 30S subunit and mRNA. The conserved region surrounding A1492 and A1493 was called the "decoding site", based on the results of chemical probing experiments and antibiotic resistance mutations. Crystallographic studies from the Ramakrishnan laboratory have now shown that G530 loop, A1492 and A1493 undergo localized conformational changes to form an RNA structure that positions these three bases to inspect the accuracy of the codon-anticodon match with high stereochemical precision, using A-minor interactions. Some results from the pre-X-ray era may provide clues to further aspects of the decoding process.     

Structural insights into eukaryotic ribosomes and the initiation of translation

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Highlights? Overview of recent progress in the structural characterization of eukaryotic ribosomes and initiation complexes. ? Crystal structures, cryo-EM and biochemical data are combined to derive structural models of larger assemblies. ? Homology models of eukaryotic initiation complexes provide a starting point for future experiments.     

Structure of a eukaryotic decoding region A-site RNA

The aminoglycoside antibiotics target a region of highly conserved nucleotides in the aminoacyl-tRNA site (A site) of 16 S RNA on the 30 S subunit. The structures of a prokaryotic decoding region A-site oligonucleotide free in solution and bound to the aminoglycosides paromomycin and gentamicin C1A have been determined. Here, the structure of a eukaryotic decoding region A-site oligonucleotide has been determined using homonuclear and heteronuclear NMR spectroscopy, and compared to the unbound prokaryotic rRNA structure. The two structures are similar, with a U1406-U1495 base-pair, a C1407-G1494 Watson-Crick base-pair, and a G1408-A1493 base-pair instead of the A1408-A1493 base-pair of the prokaryotic structure. The two structures differ in the orientation of the 1408 position with respect to A1493; G1408 is rotated toward the major groove, which is the binding pocket for aminoglycosides. The structures also differ in the stacking geometry of G1494 on A1493, which could have slight long-range conformational effects.