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At least 19 recordsLinked to original sources
PubMed1992

Loss of transfer RNA genes from the plastid 16S-23S ribosomal RNA gene spacer in a parasitic plant.

The plastid 16S-23S intergenic spacer region in Conopholis americana, a totally heterotrophic angiosperm in the family Orobanchaceae, has undergone large deletions, including the entire tRNA(Ile) gene and all but small remnants of the tRNA(Ala) gene. The length of the region is less than 20% of that of other land plants which have been investigated, making it the smallest 16S-23S intergenic spacer reported thus far for any land plant. The remaining sequences in the spacer are 90.1% identical to tobacco, indicating that, while the region is well conserved at the sequence level, it is evolving rapidly by deletion. Experiments using the polymerase chain reaction and hybridization to DNA gel blots have failed to reveal either of the two missing tRNA genes elsewhere in the Conopholis cell.

Base Sequence↗
PubMed1999

The first determination of pseudouridine residues in 23S ribosomal RNA from hyperthermophilic Archaea Sulfolobus acidocaldarius.

We describe the first identification of pseudouridine (Psi) residues in ribosomal RNA (23S rRNA) of an hyperthermophilic Archaea Sulfolobus acidocaldarius. In contrast to Eucarya rRNA, only six Psi residues were detected, which is rather close to the situation in Bacteria. However, three modified positions (Psi(2479), Psi(2535) and Psi(2550)) are unique for S. acidocaldarius. Two Psi residues at positions 2060 and 2594 are universally conserved, while one other Psi (position 2066) is also common to Eucarya. Taken together the results argue against the conservation of Psi-synthases between Archaea and Bacteria and provide a basis for the search of snoRNA-like guides for Psi formation in Archaea.

Base Sequence↗
PubMed1998

Kinetic analysis of the RNA-dependent adenosinetriphosphatase activity of DbpA, an Escherichia coli DEAD protein specific for 23S ribosomal RNA.

The adenosinetriphosphatase (ATPase) activity of the Escherichia coli DEAD protein DbpA is unusual in that it is specifically stimulated by 23S ribosomal RNA (rRNA). A coupled spectroscopic ATPase assay was used to investigate the interaction of DbpA with RNA and ATP. A 153-base fragment of domain V of 23S rRNA is kinetically identical to intact, native rRNA in activating DbpA: kcat = 600 min-1, Kapp(RNA) = 10 nM, and Km(ATP) = 120 microM. The ATPase turnover in the absence of RNA is 0.25 min-1. Fragments of 23S rRNA lacking this site (nucleotides 2454-2606) are essentially inactive, as are other RNAs such as poly(A) and tRNA. The relative RNA specificity of DbpA ranges from 10(3) to 10(6) [kmax/Kapp(RNA)]. The interaction with this small RNA fragment was further investigated with regard to stoichiometry, pH, salt and temperature. DbpA is not activated by E. coli ribosomes, nor by large subunits, while denatured ribosomes stimulate full ATPase activity. Taken together with the tight, site-specific binding to naked, unmodified 23S rRNA, this suggests a role for DbpA in ribosome biogenesis rather than translation.

Adenosine Triphosphatases↗
PubMed1996

The 23S Ribosomal RNA Mutation Database (23SMDB).

The 23S Ribosomal RNA Mutation Database (23SMDB), provides a list of mutated positions in 23S ribosomal RNA from Escherichia coli and the identity of each alteration. Information provided for each mutation includes: (i) a brief description of the phenotypes(s) associated with each mutation, (ii) whether a mutant phenotype has been detected by in vivo or in vitro methods, and (iii) relevant literature citations. The database is available via ftp and on the World Wide Web.

Computer Communication Networks↗
PubMed1998

Ribosome-catalyzed peptide-bond formation with an A-site substrate covalently linked to 23S ribosomal RNA.

In the ribosome, the aminoacyl-transfer RNA (tRNA) analog 4-thio-dT-p-C-p-puromycin crosslinks photochemically with G2553 of 23S ribosomal RNA (rRNA). This covalently linked substrate reacts with a peptidyl-tRNA analog to form a peptide bond in a peptidyl transferase-catalyzed reaction. This result places the conserved 2555 loop of 23S rRNA at the peptidyl transferase A site and suggests that peptide bond formation can occur uncoupled from movement of the A-site tRNA. Crosslink formation depends on occupancy of the P site by a tRNA carrying an intact CCA acceptor end, indicating that peptidyl-tRNA, directly or indirectly, helps to create the peptidyl transferase A site.

Anti-Bacterial Agents↗
PubMed1990

Cloning, in vitro transcription, and biological activity of Escherichia coli 23S ribosomal RNA.

The 23S rRNA gene was excised from the rrnB operon of pKK3535 and ligated into pUC19 behind the strong class III T7 promoter so that the correct 5' end of mature 23S RNA was produced upon transcription by T7 RNA polymerase. At the 3' end, generation of a restriction site for linearization required the addition of 2 adenosine residues to the mature 23S sequence. In vitro runoff transcripts were indistinguishable from natural 23S RNA in size on denaturing gels and in 5'-terminal sequence. The length and sequence of the 3' terminal T1 fragment was also as expected from the DNA sequence, except that an additional C, A, or U residue was added to 21%, 18%, or 5% of the molecules, respectively. Typical transcription reactions yielded 500-700 moles RNA per mole template. This transcript was used as a substrate for methyl transfer from S-adenosyl methionine catalyzed by Escherichia coli cell extracts. The majority (50-65%) of activity observed in a crude (S30) extract appeared in the post-ribosomal supernatant (S100). Activities catalyzing formation of m5C, m5U, m2G, and m6A residues in the synthetic transcript were observed.

Base Sequence↗
PubMed1976

Extensions of the known sequences at the 3' and 5' ends of 23S ribosomal RNA from Escherichia coli, possible base pairing between these 23S RNA regions and 16S ribosomal RNA.

Extensions of the known sequences at both 3' and 5' ends of 23S ribosomal RNA are presented: The 5' terminal is pG-G-U-U-A-A-G-Cp or pG-G-U... G-U-U-A-A-G-Cp, with a very short sequence between Up and Gp and the 3'terminal is G-A-A-C-C-G-A-(G)-G-C-U-U-A-A-C-C-U-UOH. These two terminal regions exhibit a high degree of complementarity. In addition, extensive complementarities are also found between the 5'terminal sequence of 23S RNA and a sequence contained in section A of the 16S ribosomal RNA, and between the 3'terminal sequence of 23S RNA and sequences in sections O and J in the 16S RNA. The degree of complementarity between the two extremities of 23S RNA, and between these extremities and regions of the 16S RNA, is far greater than would be expected on a random basis suggesting a possible involvement of this base-pairing in the functioning of ribosomes. This possibility is discussed.

Base Sequence↗
PubMed1998

Reconstitution of peptide bond formation with Escherichia coli 23S ribosomal RNA domains.

It was recently demonstrated that peptide bond formation can occur using an Escherichia coli naked 23S ribosomal RNA without any of the ribosomal proteins. Here, the six domains of the 23S ribosomal RNA were individually synthesized and shown to be capable, when complexed together, of stimulating the reaction. Omission and addition experiments indicated that the activity could be reconstituted solely by domain V at a concentration 10 times higher than that of the intact 23S ribosomal RNA, whereas domain VI could enhance the activity in trans. These findings suggest that fragments of an RNA molecule have the ability to associate into a functional whole.

Catalysis↗
PubMed1998

New features of 23S ribosomal RNA folding: the long helix 41-42 makes a "U-turn" inside the ribosome.

23S rRNA from Escherichia coli was cleaved at single internucleotide bonds using ribonuclease H in the presence of appropriate chimeric oligonucleotides; the individual cleavage sites were between residues 384 and 385, 867 and 868, 1045 and 1046, and 2510 and 2511, with an additional fortuitous cleavage at positions 1117 and 1118. In each case, the 3' terminus of the 5' fragment was ligated to radioactively labeled 4-thiouridine 5'-,3'-biphosphate ("psUp"), and the cleaved 23S rRNA carrying this label was reconstituted into 50S subunits. The 50S subunits were able to associate normally with 30S subunits to form 70S ribosomes. Intra-RNA crosslinks from the 4-thiouridine residues were induced by irradiation at 350 nm, and the crosslink sites within the 23S rRNA were analyzed. The rRNA molecules carrying psUp at positions 867 and 1117 showed crosslinks to nearby positions on the opposite strand of the same double helix where the cleavage was located, and no crosslinking was detected from position 2510. In contrast, the rRNA carrying psUp at position 384 showed crosslinking to nt 420 (and sometimes also to 416 and 425) in the neighboring helix in 23S rRNA, and the rRNA with psUp at position 1045 gave a crosslink to residue 993. The latter crosslink demonstrates that the long helix 41-42 of the 23S rRNA (which carries the region associated with GTPase activity) must double back on itself, forming a "U-turn" in the ribosome. This result is discussed in terms of the topography of the GTPase region in the 50S subunit, and its relation to the locations of the 5S rRNA and the peptidyl transferase center.

Base Sequence↗
PubMed2003

Prevalence of Helicobacter pylori resistance to clarithromycin and metronidazole determined by 23S ribosomal RNA and rdxA gene analyses in Hiroshima, Japan.

BACKGROUND AND AIMS: Resistance to antibiotics in Helicobacter pylori is increasing and becoming a serious problem in eradication treatment of H. pylori. The prevalence of H. pylori infections that are resistant to clarithromycin, metronidazole, or both were determined in H. pylori isolates in Hiroshima, Japan. METHODS: Sixty Japanese patients with H. pylori infection were collected between 1999 and 2000. To detect the resistance to clarithromycin and metronidazole, mutations of the 23S ribosomal RNA (rRNA) and rdxA genes that are responsible for resistance in H. pylori, were examined by direct sequencing analysis. RESULTS: Resistance to clarithromycin and metronidazole was detected in 12 (20.0%) and nine (15.0%) of the patients, respectively. Dual resistance to clarithromycin and metronidazole was detected in five (8.3%) patients. CONCLUSION: These results indicate that the relatively high prevalence of the dual resistance in H. pylori isolates may need special attention and new therapeutic approaches in Japan.

Anti-Bacterial Agents↗
PubMedDate not supplied

The binding of thiostrepton to 23S ribosomal RNA.

The antibiotic, thiostrepton, binds to 23S ribosomal RNA from E coli with a dissociation constant (KD) of 2.4 x 10(-7) M. The specificity of the interaction was established using 16S rRNA and modified or mutationally-altered 23S rRNA. Thus, no binding was detected with rRNA from the 30S subunit nor with rRNA modified in vitro by the thiostrepton resistance methylase. Mutant 23S rRNA, altered at residue 1067 in each of the 3 possible ways, showed reduced binding affinity for thiostrepton. The KD for the G mutation was 3.5 x 10(-6) M; for the C mutation, 2.4 x 10(-5) M; and for the U mutation, 4.8 x 10(-5) M. This reduction in drug binding is compatible with functional analyses; the C or U mutation results in ribosomal particles which are poorly inhibited by the drug compared with wild-type, whereas the G mutation results in an intermediate response to the drug in protein synthesis. The smallest 23S rRNA fragment used here that was capable of binding thiostrepton, in a nitrocellulose filter binding assay, comprised residues 1052-1112 and the dissociation constant was 3.0 x 10(-7) M, ie virtually indistinguishable from that with intact 23S RNA. However, the drug was incapable of binding to the 5'-moiety of this fragment (ie residues 1052-1084) or to an RNA transcript complementary to 1052-1112.

Binding Sites↗
PubMed2000

Sequence diversity of intervening sequences (IVSs) in the 23S ribosomal RNA in Salmonella spp.

Intervening sequences (IVSs) occur sporadically in the rrl (ribosomal RNA large) genes for 23S ribosomal RNA (rRNA) at helix-25 (base pair 550) and helix 45 (base pair 1170) in several bacterial genera, including Salmonella, Yersinia, Proteus, and Providencia, representing the Enterobacteriaceae, but are missing from other genera such as Escherichia. These sequences are transcribed, but later excised without re-ligation during RNaseIII processing of the rRNA, resulting in fragmented 23S rRNA. The IVSs from 22 strains of the SARB (Salmonella Reference Collection B) set were amplified by PCR and sequenced.IVSs with 90% or more sequence identity were placed in the same family; Salmonella has three families of IVSs in helix-25 (A, B, and C) and two in helix-45 (M and O). The rRNA secondary structure for the IVSs predicted from the mfold program reveals a primary stem of about 14bp, which is the postulated RNaseIII cleavage site, and a secondary region of stems and loops. The primary stem is considerably well conserved, with a high rate of compensatory mutations (positional covariants), confirming the reality of the secondary structure and indicating that removal of the IVSs exerts a positive selective pressure to retain the secondary structure. The pattern of possession and presence of families of IVSs was diverse and could not be related to the proposed ancestry of the strains as revealed by the multi-locus enzyme electrophoresis pattern of the strains, suggesting that the IVSs are transferred between strains by lateral transfer. Helix-25 IVSs from families A, B, and C of Salmonella and D of Proteus, which share almost identical primary stems, are placed in superfamily I, while the primary stems of other IVSs from Proteus and Providencia are unrelated to superfamily I and are thus placed into superfamily II; this indicates lateral transfer of members of superfamily I between Proteus and Salmonella, but an independent origin of IVSs of superfamily II in Proteus and Providencia.

Base Sequence↗
PubMed1993

Amplification and sequencing of variable regions in bacterial 23S ribosomal RNA genes with conserved primer sequences.

Published bacterial 23S ribosomal RNA sequences were aligned, and universally conserved regions flanking highly variable regions were looked for. In strategically positioned conserved regions, six oligonucleotides suitable for polymerase chain reaction (PCR) and sequencing were designed, allowing fast sequencing of four of the most variable 23S rRNA regions. Two other primers were designed for PCR amplification of nearly complete 23S rRNA genes. All these primers successfully amplified fragments of 23S rRNA genes from seven unrelated bacteria. Four primers were used to determine 938 bp of sequence for Campylobacter jejuni subsp. jejuni. These results indicate that the oligonucleotide sequences presented here are useful for PCR amplification and sequence determination of variable 23S rRNA regions for a broad variety of eubacterial species.

Base Sequence↗
PubMed1998

Expanded versions of the 16S and 23S ribosomal RNA mutation databases (16SMDBexp and 23SMDBexp)

Expanded versions of the Ribosomal RNA Mutation Databases provide lists of mutated positions in 16S and 16S-like ribosomal RNA (16SMDBexp) and 23S and 23S-like ribosomal RNA (23SMDBexp) and the identity of each alteration. Alterations from organisms other than Escherichia coli are reported at positions according to the E.coli numbering system. Information provided for each mutation includes: (i) a brief description of the phenotype(s) associated with each mutation, (ii) whether a mutant phenotype has been detected by in vivo or in vitro methods, and (iii) relevant literature citations. The databases are available via ftp and on the World Wide Web at the following URL: http: //www.fandm.edu/Departments/Biology/Databases/RNA.h tml

Computer Communication Networks↗
PubMed1999

The macrolide-ketolide antibiotic binding site is formed by structures in domains II and V of 23S ribosomal RNA.

The macrolide antibiotic erythromycin interacts with bacterial 23S ribosomal RNA (rRNA) making contacts that are limited to hairpin 35 in domain II of the rRNA and to the peptidyl transferase loop in domain V. These two regions are probably folded close together in the 23S rRNA tertiary structure and form a binding pocket for macrolides and other drug types. Erythromycin has been derivatized by replacing the L-cladinose moiety at position 3 by a keto group (forming the ketolide antibiotics) and by an alkyl-aryl extension at positions 11/12 of the lactone ring. All the drugs footprint identically within the peptidyl transferase loop, giving protection against chemical modification at A2058, A2059 and G2505, and enhancing the accessibility of A2062. However, the ketolide derivatives bind to ribosomes with widely varying affinities compared with erythromycin. This variation correlates with differences in the hairpin 35 footprints. Erythromycin enhances the modification at position A752. Removal of cladinose lowers drug binding 70-fold, with concomitant loss of the A752 footprint. However, the 11/12 extension strengthens binding 10-fold, and position A752 becomes protected. These findings indicate how drug derivatization can improve the inhibition of bacteria that have macrolide resistance conferred by changes in the peptidyl transferase loop.

Anti-Bacterial Agents↗
PubMed1994

Overexpression of the thiostrepton-resistance gene from Streptomyces azureus in Escherichia coli and characterization of recognition sites of the 23S rRNA A1067 2'-methyltransferase in the guanosine triphosphatase center of 23S ribosomal RNA.

The thiostrepton-resistance gene encoding the 23S rRNA A1067 methyltransferase from Streptomyces azureus has been overexpressed in Escherichia coli using a T7-RNA-polymerase-dependent expression vector. The protein was efficiently expressed at levels up to 20% of total soluble protein and purified to near homogeneity. Kinetic parameters for S-adenosyl-L-methionine (Km = 0.1 mM) and an RNA fragment containing nucleotides 1029-1122 of the 23S ribosomal RNA from E. coli (Km = 0.001 mM) were determined. S-Adenosyl-L-homocysteine showed competitive product inhibition (Ki = 0.013 mM). Binding of either thiostrepton or protein L11 inhibited methylation. RNA sequence variants of the RNA fragment with mutations in nucleotides 1051-1108 were tested as substrates for the methylase. The experimental data indicate that methylation is dependent on the secondary structure of the hairpin including nucleotide A1067 and the exact sequence U(1066)-A(1067)-G(1068)-A(1069)-A(1070) of the single strand.

Base Sequence↗
PubMed2000

Translational defects of Escherichia coli mutants deficient in the Um(2552) 23S ribosomal RNA methyltransferase RrmJ/FTSJ.

We recently identified RrmJ (alias FtsJ), the first encoded protein of the rrmJ-hflB heat shock operon, as an Um(2552) methyltransferase of the 23S rRNA. We now report that the rrmJ-deficient strain exhibits growth and translational defects compared to the wild-type strain. Growth rates of the rrmJ mutant are decreased at both low and high temperatures. Protein synthesis activity is reduced up to 65% when S(30) rrmJ mutant extracts are tested in a coupled in vitro transcription/translation assay. In vitro methylation of these extracts by RrmJ partially restores protein synthesis activity. Polysome profile analysis of the rrmJ strain reveals an increase in the proportion of free 30S and 50S subunits at both 30 and 42 degrees C. These results suggest that the RrmJ-catalyzed methylation of Um(2552) in 23S RNA strengthens ribosomal subunit interactions, increases protein synthesis activity, and improves cell growth rates even at non-heat shock temperatures.

Anti-Bacterial Agents↗