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PubMed2005

Effects of nucleotide substitution and modification on the stability and structure of helix 69 from 28S rRNA.

The helix 69 (H69) region of the large subunit (28S) rRNA of Homo sapiens contains five pseudouridine (Psi) residues out of 19 total nucleotides (26%), three of which are universally or highly conserved. In this study, the effects of this abundant modified nucleotide on the structure and stability of H69 were compared with those of uridine. The role of a loop nucleotide substitution from A in bacteria (position 1918 in Escherichia coli 23S rRNA) to G in eukaryotes (position in 3734 in H. sapiens) was also examined. The thermodynamic parameters were obtained through UV melting studies, and differences in the modified and unmodified RNA structures were examined by 1H NMR and circular dichroism spectroscopy. In addition, a [1,3-15N]Psi phosphoramidite was used to generate H69 analogs with site-specific 15N labels. By using this approach, different Psi residues can be clearly distinguished from one another in 1H NMR experiments. The effects of pseudouridine on H. sapiens H69 are consistent with previous studies on tRNA, rRNA, and snRNA models in which the nucleotide offers stabilization of duplex regions through PsiN1H-mediated hydrogen bonds. The overall secondary structure and base-pairing patterns of human H69 are similar to the bacterial RNA, consistent with the idea that ribosome structure and function are highly conserved. Nonetheless, pseudouridine-containing RNAs have subtle differences in their structures and stabilities compared to the corresponding uridine-containing analogs, suggesting possible roles for Psi such as maintaining translation fidelity.

Circular Dichroism↗
PubMed2004

Comparison of tetracycline and tigecycline binding to ribosomes mapped by dimethylsulphate and drug-directed Fe2+ cleavage of 16S rRNA.

OBJECTIVES: The new antibiotic tigecycline (9-t-butylglycylamido-minocycline; GAR-936) overcomes most of the known tetracycline resistance mechanisms. Here we analyse its mode of antibiotic action by probing 70S ribosomes of Escherichia coli with dimethylsulphate (DMS) and Fe(2+)-mediated cleavage to identify binding sites of tetracycline and tigecycline. METHODS: Fe(2+)-mediated cleavage makes use of the ability of Fe2+ to replace the Mg2+ ion complexed with tetracyclines. After addition of H2O2, Fe2+ generates short-lived, highly reactive hydroxyl radicals that can cleave RNA close to the tetracycline binding sites. RESULTS: We identified three prominent Fe(2+)-mediated cleavage sites in helices 29 and 34, and in the internal loop of helix 31 of 16S rRNA in the presence of tetracycline or tigecycline. Qualitatively, these sites are modified identically by both antibiotics, but quantitative differences observed in the cleavage intensities indicate that the drugs bind in slightly different orientations. These results are supported by DMS modification, mutational analysis of 16S rRNA and structural modelling of tigecycline at a tetracycline-binding site in the 30S ribosomal subunit. CONCLUSIONS: Both derivatives bind to identical or overlapping sites and probably share the same mode of antibiotic action. The fact that tigecycline overcomes most of the known tetracycline resistance mechanisms is interpreted as a result of steric hindrance due to the large substituent at position 9.

Base Sequence↗
PubMed2001

Interactions between 23S rRNA and tRNA in the ribosomal E site.

Interactions between tRNA or its analogs and 23S rRNA in the large ribosomal subunit were analyzed by RNA footprinting and by modification-interference selection. In the E site, tRNA protected bases G2112, A2392, and C2394 of 23S rRNA. Truncated tRNA, lacking the anticodon stem-loop, protected A2392 and C2394, but not G2112, and tRNA derivatives with a shortened 3' end protected only G2112, but not A2392 or C2394. Modification interference revealed C2394 as the only accessible nucleotide in 23S rRNA whose modification interferes with binding of tRNA in the large ribosomal subunit E site. The results suggest a direct contact between A76 of tRNA A76 and C2394 of 23S rRNA. Protections at G2112 may reflect interaction of this 23S rRNA region with the tRNA central fold.

Anticodon↗
PubMed2004

Spb1p-directed formation of Gm2922 in the ribosome catalytic center occurs at a late processing stage.

rRNA molecules undergo extensive posttranscriptional modification, predominantly 2'-O-ribose methylation and pseudouridine formation, both of which are guided by the numerous small nucleolar RNAs in eukaryotes. Here, we describe an exception to this rule. The essential yeast nucleolar protein Spb1p is a site-specific rRNA methyltransferase modifying the universally conserved G2922 that is located within the A loop of the catalytic center of the ribosome. The equivalent position in bacteria is the docking site for aminoacyl-tRNA, and it is critical for translation. In sharp contrast to other 2'-O-methylriboses that are formed on the primary transcript, Gm2922 appears at a late processing stage, during the maturation of the 27S pre-rRNA. Thus, eukaryotes have maintained a site-specific enzyme to catalyze the methylation of a nucleotide that plays a crucial role in ribosome biogenesis and translation.

Amino Acid Motifs↗
PubMed1990

[The use of RNAse H for digestion of alkylated 16S rRNA at the binding sites of addressed reagents--oligodeoxyribonucleotide derivatives].

It is demonstrated that 16S rRNA, complementary-addressed labelled with 2',3'-O-[4-N-methyl-N-(2-chloroethyl)-amino]benzylidene derivatives of oligonucleotides d(pACCTTGTT)rA and d(pTTTGCTCCCC)rA, can be cleaved by RNase H within the adducts, resulted from the modification. Comparative study of the 16S rRNA cleavage with RNase H within the above--mentioned covalent adducts, on the one hand, and within heteroduplexes with the same oligodeoxyribonucleotides, on the other, showed that(i) the complementary-addressed modification proceeds both in perfect and non-per ect complexes; (ii) 16S rRNA is cleaved by RNase H within both perfect and non-perfect complexes resulted from the alkylation, non-perfect complexes being considerably stabilized by the covalent bond between the reagent and the RNA; (iii) non-perfect complexes of 16S rRNA with the free oligodeoxyribonucleotides are unstable even at the high oligonucleotide concentration, so that no cleavage of 16S rRNA in such duplexes is observed. The approach based on cleavage of RNA within covalent adducts resulted from the complementary-addressed RNA modification may be used for fragmentation of RNA molecule in the addressed reagent's binding site.

Alkylation↗
PubMed1969

Differential stability of 28s and 18s rat liver ribosomal ribonucleic acids.

Rat liver ribosomal RNA (rRNA) free from nuclease contaminants was isolated by a modification of the phenol technique. The 28s and 18s rRNA species were separated by preparative agar-gel electrophoresis. The two rRNA species were heated at different temperatures under various conditions and the amount of undegraded rRNA was determined by analytical agar-gel electrophoresis. The 18s rRNA remained unaltered after heating for up to 10min. at 90 degrees in water, acetate buffer, pH5.0, or phosphate buffer, pH7.0. Under similar or milder conditions 28s rRNA was partially degraded, giving rise to a well-delimited 6s peak and a heterogeneous material located in the zone between 28s and 6s. The dependence of degradation of 28s rRNA on the temperature and the ionic strength of the medium was studied. The greatest extent of degradation of 28s rRNA was observed on heating at 90 degrees in water. It is suggested that the instability of rat liver 28s rRNA is due to two factors: the presence of hidden breaks in the polymer chain and a higher susceptibility of some phosphodiester bonds to thermal hydrolysis.

Animals↗
PubMed2004

Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure.

A dynamic programming algorithm for prediction of RNA secondary structure has been revised to accommodate folding constraints determined by chemical modification and to include free energy increments for coaxial stacking of helices when they are either adjacent or separated by a single mismatch. Furthermore, free energy parameters are revised to account for recent experimental results for terminal mismatches and hairpin, bulge, internal, and multibranch loops. To demonstrate the applicability of this method, in vivo modification was performed on 5S rRNA in both Escherichia coli and Candida albicans with 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate, dimethyl sulfate, and kethoxal. The percentage of known base pairs in the predicted structure increased from 26.3% to 86.8% for the E. coli sequence by using modification constraints. For C. albicans, the accuracy remained 87.5% both with and without modification data. On average, for these sequences and a set of 14 sequences with known secondary structure and chemical modification data taken from the literature, accuracy improves from 67% to 76%. This enhancement primarily reflects improvement for three sequences that are predicted with <40% accuracy on the basis of energetics alone. For these sequences, inclusion of chemical modification constraints improves the average accuracy from 28% to 78%. For the 11 sequences with <6% pseudoknotted base pairs, structures predicted with constraints from chemical modification contain on average 84% of known canonical base pairs.

Algorithms↗
PubMed1985

Electron microscopy of the secondary structure in partially denatured rRNAs of Escherichia coli and Bacillus stearothermophilus.

Partially denatured 16S and 23S rRNAs from the thermophile Bacillus stearothermophilus show characteristic loop patterns when observed by electron microscopy. The patterns are very similar to those seen in rRNAs from Escherichia coli. At least 2 of 4 most stable interactions in 16S rRNA and 8 of 12 interactions in 23S rRNA are in common for the two species. These interactions correspond well to features of secondary structure in models inferred for rRNA from phylogenetic sequence comparisons and chemical modification studies. However, two additional large loops, enclosing large portions of the 23S rRNA, have been detected in B. stearothermophilus for the first time, and even though other loops are similar, their relative frequencies vary in the two species. Much of the variation is consistent with relative delta G degree values for putative base-paired stems at the base of different loops; but the 5'-terminal loops in 23S rRNA, for example, are unaccountably far less stable in B. stearothermophilus. Also, in general, structural features are not differentially stabilized in B. stearothermophilus; the relative stability of secondary structure in its ribosomes at elevated growth temperatures must involve interactions with ribosomal proteins or other cellular components.

Escherichia coli↗
PubMed1997

Function and synthesis of small nucleolar RNAs.

Eukaryotic cells contain an extraordinarily complex population of small nucleolar RNAs (snoRNAs). During its brief lifetime, each human pre-rRNA molecule will transiently associate with approximately 150 different snoRNA species. In the past year our understanding of snoRNAs has been clarified by the recognition that the snoRNA population can be divided into a small number of groups which are structurally and functionally distinct. The two largest groups of snoRNAs direct the site-specific modification of the pre-rRNA at positions of 2'-O-methylation and pseudouridine formatio. Other groups of snoRNAs function in pre-rRNA cleavage and in the formation of the correct structure of the pre-rRNA.

Animals↗
PubMed1984

Probing the conformation of 18S rRNA in yeast 40S ribosomal subunits with kethoxal.

Yeast 40S ribosomal subunits have been reacted with kethoxal to probe the conformation of 18S rRNA. Over 130 oligonucleotides were isolated by diagonal electrophoresis and sequenced, allowing identification of 48 kethoxal-reactive sites in the 18S rRNA chain. These results generally support a secondary structure model for 18S rRNA derived from comparative sequence analysis. Significant reactivity at positions 1436 and 1439, in a region shown to be base paired by comparative analysis, lends support to the earlier suggestion [Chapman, N.M., & Noller, H.F. (1977) J. Mol. Biol 109, 131-149] that part of the 3'-major domain of 16S-like rRNAs may undergo a biologically significant conformational rearrangement. Modification of positions in 18S rRNA analogous to those previously found for Escherichia coli 16S rRNA argues for extensive structural homology between 30S and 40S ribosomal subunits, particularly in regions thought to be directly involved in translation.

Aldehydes↗
PubMed1997

Epigenetic silencing of RNA polymerase I transcription: a role for DNA methylation and histone modification in nucleolar dominance.

Nucleolar dominance is an epigenetic phenomenon that describes nucleolus formation around rRNA genes inherited from only one progenitor of an interspecific hybrid or allopolyploid. The phenomenon is widespread, occurring in plants, insects, amphibians, and mammals, yet its molecular basis remains unclear. We have demonstrated nucleolar dominance in three allotetraploids of the plant genus Brassica. In Brassica napus, accurately initiated pre-rRNA transcripts from one progenitor, Brassica rapa are detected readily, whereas transcripts from the approximately 3000 rRNA genes inherited from the other progenitor, Brassica oleracea, are undetectable. Nuclear run-on confirmed that dominance is controlled at the level of transcription. Growth of B. napus seedlings on 5-aza-2'-deoxycytidine to inhibit cytosine methylation caused the normally silent, under-dominant B. oleracea rRNA genes to become expressed to high levels. The histone deacetylase inhibitors sodium butyrate and trichostatin A also derepressed silent rRNA genes. These results reveal an enforcement mechanism for nucleolar dominance in which DNA methylation and histone modifications combine to regulate rRNA gene loci spanning tens of megabase pairs of DNA.

Azacitidine↗
PubMed1993

Erythromycin binding is reduced in ribosomes with conformational alterations in the 23 S rRNA peptidyl transferase loop.

The antibiotic erythromycin inhibits protein synthesis by binding to the 50 S ribosomal subunit, where the drug interacts with the unpaired bases 2058A and 2059A in the peptidyl transferase loop of 23 S rRNA. We used a chemical modification approach to analyse conformational changes that are induced by mutations in the peptidyl transferase loop, and to determine how these changes affect drug interaction. Mutations at positions 2057 (G-->A) and 2058 (A-->G, or -->U), all of which confer drug resistance, induce a more open conformation in the peptidyl transferase loop. Erythromycin still protects against chemical modification in the mutant peptidyl transferase loops, but the affinity of the drug interaction is reduced 20-fold in the 2057A mutant, 10(3)-fold in the 2058U mutant and 10(4)-fold in the 2058G mutant. Single mutations at position 2032 in the adjacent hairpin loop, which have previously been shown to alter drug tolerances, gave no detectable effects on the structure of the peptidyl transferase loop or on erythromycin binding. Dual mutations at positions 2032 and 2058, however, induce a marked change in the rRNA conformation with opening of the phylogenetically conserved base-pair 2063C.2447G, and confer a slow growth, drug-sensitive phenotype. The data suggest that the target site of erythromycin lies within the peptidyl transferase loop, and that limited disruption of the conformation of this site reduces drug binding, and consequently confers resistance. In addition, there is structurally and functionally important interaction between the drug target site in the peptidyl transferase loop and position 2032.

Base Sequence↗
PubMedDate not supplied

Physical mapping of rRNA genes in Medicago sativa and M. glomerata by fluorescent in situ hybridization.

Fluorescent in situ hybridization (FISH) was applied to diploid and tetraploid subspecies of alfalfa (Medicago sativa L.) to investigate the distribution of rRNA genes and to utilize the sites of 18S-5.8S-25S rDNA and 5S rDNA sequences as markers for studying the genome evolution within the species. Medicago glomerata Balb., the species considered to be the ancestor of alfalfa, was included in this study in order to obtain more information on the phylogenetics of alfalfa. Simultaneous in situ hybridization was performed with the probes pTa71 and pXVI labeled with digoxigenin and biotin, respectively. In the diploid taxa, M. glomerata, M. sativa ssp. coerulea Schmalh and ssp. falcata Arcangeli, the 18S-5.8S-25S rDNA sequences were mapped to two sites corresponding to the secondary constrictions of the nucleolar chromosome pair, while 5S rDNA appeared to be distributed in two pairs of sites. Chromosomes carrying 5S loci could be distinguished on the basis of their morphological characteristics. The number of rDNA sites detected in the tetraploid M. sativa ssp. falcata and ssp. sativa (L.) L. & L. were twice the number found in the respective diploid ssp. falcata and ssp. coerulea. The results of this study show that the distribution of ribosomal genes was maintained during the evolutionary steps from the primitive diploid to the cultivated alfalfa. Modifications of the number of rRNA loci were not observed. The importance of in situ hybridization for improving karyotype analysis in M. sativa L. is discussed.

Genes, rRNA↗
PubMed1990

Isolation of Chinese hamster ovary ribosomal mutants differentially resistant to ricin, abrin, and modeccin.

The molecular action of ricin A chain involves cleavage of the N-glycosidic bond between ribose and the adenine 4324 nucleotides from the 5' end of mammalian 28 S rRNA (Endo, Y., and Tsurugi, K. (1987) J. Biol. Chem. 262, 8128-8130). In this paper, four ricin- and abrin-resistant Chinese hamster ovary cell mutants that possess ribosomes resistant to this N-glycosidase action are described. Three of the mutant phenotypes, Lec26, Lec27, and Lec28, were recessive in somatic cell hybrids and define at least two new lectin-resistant complementation groups. The most extensively characterized mutant type, LEC17, was dominant in such hybrids. None of the mutants were cross-resistant to modeccin. Post-mitochondrial supernatants from each of the four mutants were resistant to inhibition of cell-free protein synthesis by ricin, ricin A chain, and abrin. In addition, polysomes isolated from mutant cells were resistant to cleavage of the adenine-ribose N-glycosidic bond by ricin A chain or abrin, as assayed by the release of an approximately 470-nucleotide fragment following aniline treatment of ribosomal RNA extracted from toxin-treated polysomes. The unique lectin-resistance properties of the different mutants suggests that the accessibility of adenine 4324 to each toxin differs. It seems likely that the recessive Chinese hamster ovary ribosomal mutants reflect structural changes in different ribosomal proteins while the dominant phenotype may be due to the modification of protein(s) or rRNA involved in toxin-ribosome interaction. Further analysis of these cell lines should provide new insights into the structure/function relationships of eukaryotic ribosomes.

Abrin↗
PubMed1996

Interaction of the Bacillus stearothermophilus ribosomal protein S15 with 16 S rRNA: II. Specificity determinants of RNA-protein recognition.

S15 is a primary ribosomal protein that interacts specifically with a three-way junction in the central domain of 16 S rRNA, whose binding induces a conformational change in the RNA. In the accompanying paper, we demonstrated that S15 binds with high affinity to a 61 nucleotide RNA corresponding to the minimal rRNA binding site. Here, the sequence and structural determinants for the RNA in the Bacillus stearothermophilus S15-rRNA interaction have been probed using site-directed mutagenesis, chemical modification interference, and iodine footprinting of phosphorothioate RNA. Mutations and RNA modifications that interfere with protein binding cluster in two distinct regions, one containing an internal loop and the other containing a three-way junction. The internal loop, defined by two A.G base-pairs and a bulged guanosine, is not important for the specific interaction, however, BS15 interacts with a phylogenetically conserved G.U base-pair above this internal loop. Near the three-way junction in helix 22, a bulged adenosine and two base-pairs adjacent to the junction also provide important determinants for BS15 binding. Chemical modification interference also suggests that four highly phylogenetically conserved nucleotides in the three-way junction may form non-canonical G.G and U.A base-pairs that are required for the BS15-rRNA interaction. Ethylation modification interference suggests that BS15 binding is accompanied by a conformational change in the RNA involving orientation of helices 20 and 22 at an acute angle with respect to one another. Projection of the data provided by mutagenesis, chemical modification interference analysis, and iodine footprinting onto a three-dimensional model illustrates that BS15 is likely to interact with the minor groove along an extended face of helix 22.

Base Sequence↗
PubMed2004

Mapping of the second tetracycline binding site on the ribosomal small subunit of E.coli.

Tetracycline blocks stable binding of aminoacyl-tRNA to the bacterial ribosomal A-site. Various tetracycline binding sites have been identified in crystals of the 30S ribosomal small subunit of Thermus thermophilus. Here we describe a direct photo- affinity modification of the ribosomal small subunits of Escherichia coli with 7-[3H]-tetracycline. To select for specific interactions, an excess of the 30S subunits over tetracycline has been used. Primer extension analysis of the 16S rRNA revealed two sites of the modifications: C936 and C948. Considering available data on tetracycline interactions with the prokaryotic 30S subunits, including the presented data (E.coli), X-ray data (T.thermophilus) and genetic data (Helicobacter pylori, E.coli), a second high affinity tetracycline binding site is proposed within the 3'-major domain of the 16S rRNA, in addition to the A-site related tetracycline binding site.

Anti-Bacterial Agents↗
PubMedDate not supplied

Initiation and regulation mechanisms of ribosomal RNA transcription in the eukaryote Acanthamoeba castellanii.

Acanthamoeba rRNA transcription involves the binding of a transcription initiation factor (TIF) to the core promoter of rDNA to form the preinitiation complex. This complex is formed in the absence of RNA polymerase I, and persists for multiple rounds of initiation. Polymerase I next binds to form the initiation complex. This binding is DNA sequence-independent, and is directed by protein-protein contacts with TIF. DNA melting occurs in a separate step. In contrast to most prokaryotic transcription, melting occurs only following nucleotide addition and beta-gamma hydrolysis of ATP is not required as for polymerase II. Growth-dependent regulation of rRNA transcription is accomplished by modification of RNA polymerase I. The inactive form of polymerase (PolE) is unable to bind to the promoter and has altered heat stability. PolE is still active in elongation; thus, the modification affects the polymerase site involved in TIF contact. Modification of a polymerases I and III common subunit has been detected leading to the suggestion that transcription of stable RNAs of the ribosome might be co-regulated by this mechanism.

Acanthamoeba↗
PubMed1983

Chemical modification of guanine residues of mouse 5 S ribosomal RNA with kethoxal. (Nucleosides and nucleotides 46).

Chemical modification of mouse 5 S rRNA with kethoxal was carried out to examine the secondary structure. The guanine residues located at positions 37, 41, 56, 66, 75 and 89 were modified. The relative rates of reaction are in the order G37, G56, G89, G66, G41, G75 at 28 degrees C and G37, G41, G56, G89, G75, G66 at 35 degrees C. These results support a secondary structure model containing 5 helices and 5 loops and indicate that the region around position 37 is the most exposed in higher-order structure.

Aldehydes↗