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At least 19 recordsLinked to original sources
PubMedDate not supplied

[Degradation of messenger RNA, ribosomal RNA and 2-5A-dependent inhibition of protein biosynthesis].

In rabbit reticulocyte lysates the addition of exogenous 2-5A leads after 10-20 minutes to the inhibition of protein synthesis. This inhibition can be blocked by rat antiserum to 2-5A. In intact ribosomes the ribosomal RNA is cleaved after 2-5A addition, but this cleavage is not in correlation with the protein synthesis shutoff. Ribosomal 5S RNA and 5,8S RNA are not cleaved even after several hours of incubation with 2-5A. The degradation of polysome associated mRNA correlates with the protein synthesis inhibition as revealed by Northern blot hybridization of globin mRNA with 32P-labelled p beta G plasmid. The addition of 2-5A antiserum to the rabbit reticulocyte lysate also inhibits the degradation of polysome bound globin mRNA.

2',5'-Oligoadenylate Synthetase↗
PubMed1983

Determination of the promoter strength in the mixed transcription system. II. Promoters of ribosomal RNA, ribosomal protein S1 and recA protein operons from Escherichia coli.

Using the in vitro mixed transcription system (Kajitani, M. and Ishihama, A. (1983) Nucleic Acids Res. 11, 671-686), we determined the two parameters of the promoter strength, i.e., the rate of open complex formation between RNA polymerase and promoter, and the saturation level of the open complex formation at equilibrium, for the promoters of ribosomal RNA (rrnE), ribosomal protein S1 (rpsA) and recA protein (recA) operons from Escherichia coli. Taken together with the previous determinations for lactose (lac(UV5)), tryptophan (trp) and ribosomal protein L10 (rp1J) operons, these studies revealed that the relative promoter strengths with respect to the kinetic parameter are 200, 70, 50, 40, 30, 20 and 2% of the reference promoter lacP(UV5) for recAp, rp1Jp, rpsAp3, trpP, rpsAp1, rrnEp1 and rrnEp2, respectively, under our standard reaction conditions (50 mM NaCl and 37 degrees C); and those with respect to the thermodynamic parameter are 70, 35, 20, 10, 10, 10 and 5% the level of lacP(UV5) for rrnEp2, trpP, rpsAp3, rp1Jp, rpsAp1, rrnEp1 and recAp, respectively. The order of the promoter strength, however, changes with variation of the salt concentration or reaction temperature.

Bacterial Proteins↗
PubMed1976

Rates of synthesis of polyadenylated messenger RNA and ribosomal RNA during the cell cycle of Schizosaccharomyces pombe. With an appendix: calculation of the pattern of protein accumulation from observed changes in the rate of messenger RNA synthesis.

The rates of polyadenylated messenger RNA and ribosomal RNA synthesis were measured in synchronously dividing cultures of fission yeast (Schizosaccharomyces pombe). Control asynchronous cultures, which had been exposed to the conditions used for preparing synchronous cultures, were investigated to check for effects of the synchronization procedure itself on RNA synthesis. After each period of DNA synthesis in synchronous culture, the rates of messenger and ribosomal RNA synthesis doubled, suggesting that gene number controls the rate of messenger and ribosomal RNA synthesis. This was confirmed by experiments with asynchronous, exponential-phase cultures in which DNA synthesis was inhibited by hydroxyurea. Both synchronous culture and hydroxyurea experiments suggested that there is a delay of 15 min (0-1 of the cell generation time) between replication of the DNA and transcription of both gene copies. A pattern of protein accumulation was calculated from changes in the rate of polyadenylated messenger RNA synthesis during synchronous culture. The simulated pattern indicates that protein is accumulated linearly, with a doubling in the rate of accumulation once per cell cycle. The simulated pattern of protein accumulation is very similar to measurements previously reported by other workers of changes in activities of 3 enzymes in synchronous cultures. It is suggested that the doubling of the rate of messenger RNA synthesis, as a consequence of the replication of the DNA once per cycle, provides the basis of a mechanism for control of the doubling of other cellular constituents during the cell cycle.

Adenine↗
PubMed1998

Directed hydroxyl radical probing of 16S ribosomal RNA in ribosomes containing Fe(II) tethered to ribosomal protein S20.

The 16S ribosomal RNA neighborhood of ribosomal protein S20 has been mapped, in both 30S subunits and 70S ribosomes, using directed hydroxyl radical probing. Cysteine residues were introduced at amino acid positions 14, 23, 49, and 57 of S20, and used for tethering 1-(p-bromoacetamidobenzyl)-Fe(II)-EDTA. In vitro reconstitution using Fe(II)-derivatized S20, together with the remaining small subunit ribosomal proteins and 16S ribosomal RNA (rRNA), yielded functional 30S subunits. Both 30S subunits and 70S ribosomes containing Fe(II)-S20 were purified and hydroxyl radicals were generated from the tethered Fe(II). Hydroxyl radical cleavage of the 16S rRNA backbone was monitored by primer extension. Different cleavage patterns in 16S rRNA were observed from Fe(II) tethered to each of the four positions, and these patterns were not significantly different in 30S and 70S ribosomes. Cleavage sites were mapped to positions 160-200, 320, and 340-350 in the 5' domain, and to positions 1427-1430 and 1439-1458 in the distal end of the penultimate stem of 16S rRNA, placing these regions near each other in three dimensions. These results are consistent with previous footprinting data that localized S20 near these 16S rRNA elements, providing evidence that S20, like S17, is located near the bottom of the 30S subunit.

Base Sequence↗
PubMed1991

A study on postmortem stability of vasopressin messenger RNA in rat brain compared with those in total RNA and ribosomal RNA.

Postmortem stability of arginine-vasopressin (AVP) messenger RNA (mRNA) in the rat brain was studied comparing changes with those in the recovered amounts of total RNA and ribosomal RNA (rRNA). The amount of AVP mRNA and rRNA showed a decrease with increasing time interval after death (postmortem time), whereas the amount of total RNA did not alter with postmortem time. The half-life of AVP mRNA in the rat postmortem seemed to be approximately 16 hrs. The analysis of the ratio of AVP mRNA to 18S-rRNA suggested that AVP mRNA was degraded postmortem more rapidly than rRNA. These results suggest that autopsied human brains should be used for AVP mRNA study within a short postmortem time.

Animals↗
PubMed1983

Reverse transcription of yeast double-stranded RNA and ribosomal RNA using synthetic oligonucleotide primers.

The ability of the four oligodeoxyribonucleotide primers oligo(dT)12-18, oligo(dA)12-18, oligo(dG)12-18 and oligo(dC)12-18 to act as primers for avian myeloblastosis virus reverse transcriptase on denatured yeast double-stranded (ds) RNA templates was investigated. Oligo(dT) and oligo(dA) were found to prime the synthesis of 1.1 and 1.0 kb reverse transcripts, respectively, using denatured M dsRNA as a template. The oligo(dT)- and oligo(dA)-primed cDNAs of M dsRNA hybridized to the region of the M dsRNA that encoded the killer toxin and to each other. Addition of oligo(dT) to reverse transcription reactions of denatured L dsRNA produced a 4.3 kb cDNA. During the course of this investigation oligo(dC) was observed to be a highly efficient primer for reverse transcription of yeast 18 S ribosomal RNA. Oligo(dC) primed the synthesis of a 1.0 kb transcript of 18 S rRNA which hybridized to the large Eco RI fragment of the 18 S rRNA gene. Reverse transcription of double-stranded RNA and 25 S ribosomal RNA was found to occur to some extent in the absence of added oligonucleotide primer.

Avian Myeloblastosis Virus↗
PubMed1991

Recognition of the highly conserved GTPase center of 23 S ribosomal RNA by ribosomal protein L11 and the antibiotic thiostrepton.

The antibiotic thiostrepton, a thiazole-containing peptide, inhibits translation and ribosomal GTPase activity by binding directly to a limited and highly conserved region of the large subunit ribosomal RNA termed the GTPase center. We have previously used a filter binding assay to examine the binding of ribosomal protein L11 to a set of ribosomal RNA fragments encompassing the Escherichia coli GTPase center sequence. We show here that thiostrepton binding to the same RNA fragments can also be detected in a filter binding assay. Binding is relatively independent of monovalent salt concentration and temperature but requires a minimum Mg2+ concentration of about 0.5 mM. To help determine the RNA features recognized by L11 and thiostrepton, a set of over 40 RNA sequence variants was prepared which, taken together, change every nucleotide within the 1051 to 1108 recognition domain while preserving the known secondary structure of the RNA. Binding constants for L11 and thiostrepton interaction with these RNAs were measured. Only a small number of sequence variants had more than fivefold effects on L11 binding affinities, and most of these were clustered around a junction of helical segments. These same mutants had similar effects on thiostrepton binding, but more than half of the other sequence changes substantially reduced thiostrepton binding. On the basis of these data and chemical modification studies of this RNA domain in the literature, we propose that L11 makes few, if any, contacts with RNA bases, but recognizes the three-dimensional conformation of the RNA backbone. We also argue from the data that thiostrepton is probably sensitive to small changes in RNA conformation. The results are discussed in terms of a model in which conformational flexibility of the GTPase center RNA is functionally important during the ribosome elongation cycle.

Base Composition↗
PubMed1989

Comparison of transfer RNA and ribosomal RNA intron splicing in the extreme thermophile and archaebacterium Desulfurococcus mobilis.

The structure of the exon-intron boundary was compared for an intron within 23S ribosomal RNA of Desulfurococcus mobilis and a newly discovered intron in tRNA(Met) from the same organism. The occurrence of a putative common structural feature suggests that intron excision occurs by the same mechanism. The possible recognition of this structural feature by the cleavage enzyme was investigated for the ribosomal RNA intron using RNA substrates exhibiting various exon and intron deletions. The results support the involvement of the structural features in the cleavage process. The evolutionary implications of these results are considered.

Archaea↗
PubMed2000

5S ribosomal RNA.

Ribosomes have been visualized in electron micrographs in 1943 but 5S rRNA was discovered 20 years later. The next four decades witnessed big advances in our understanding of the ribosome using biochemical, genetic and low resolution structural approaches. During those times many experimental data accumulates also on 5S rRNA, but its precise function remains unknown. To understand the role of this RNA in ribosome a high-resolution structure is urgently needed. Because the ribosome is a dynamic machine, details on the interaction of 5S rRNA with proteins within entire ribosome are required. Big progress in the structural analysis of ribosome will stimulate further understanding of 5S rRNA.

Animals↗
PubMed1984

Effects of site-directed mutations in the central domain of 16 S ribosomal RNA upon ribosomal protein binding, RNA processing and 30 S subunit assembly.

Using a multicopy plasmid encoding the Escherichia coli rrnB ribosomal RNA operon and the techniques of in vitro site-directed mutagenesis, we have introduced several small alterations into the central domain of 16 S rRNA, which encompasses nucleotides 560 to 890. Four of the rRNAs studied contained deletions and one contained an insertion. The altered small ribosomal subunit rRNAs were used to investigate relationships among 16 S rRNA processing, protein-16 S rRNA interactions and assembly of the 30 S ribosomal subunit. Analysis of plasmid-coded transcripts from maxicells revealed that products from wild-type 16 S rRNA genes were fully processed and assembled into mature 30 S subunits. Under the same conditions, the processing and assembly of transcripts derived from the mutant plasmids were severely impaired. In some instances, the mutations completely blocked both processes, while in other cases rRNA maturation and ribosome assembly were retarded, but not eliminated completely. In all cases, the mutations led to the accumulation of the 17 S precursor to 16 S rRNA. The mutant 17 S rRNAs were purified and incubated with various combinations of E. coli ribosomal proteins S6, S8, S15 and S18, which are known to bind to the central domain of 16 S rRNA. Ribonuclease digestion of the resulting protein-17 S rRNA complexes and fractionation of the products permitted detection of three distinct protein-RNA fragment complexes which contained S8, S8 + S15, or S6 + S8 + S15 + S18. Whereas wild-type 17 S rRNA was able to form all three of these complexes, deletion of nucleotides 693 to 721 or 822 to 874 abolished the interaction of S6 and S18, and removal of nucleotides 659 to 718 prevented the binding of S6, S15 and S18. In contrast, elimination of residue 614, or the presence of a 16-base insertion between nucleotides 614 and 615, had no significant effect on the binding of any of the four proteins tested. Together, our results demonstrate that 16 S rRNA maturation and 30 S subunit assembly are tightly coupled, and show that, in at least some cases, defects in these processes can be correlated with the inability of particular ribosomal proteins to associate with altered rRNA molecules. Moreover, we have confirmed the essentiality of certain rRNA sequences for the formation and/or stabilization of these protein-rRNA interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Centrifugation, Density Gradient↗
PubMed1986

The binding of the antitumor antibiotic chartreusin to poly(dA-dT).poly(dA-dT), poly(dG-dC).poly(dG-dC), calf thymus DNA, transfer RNA, and ribosomal RNA.

Chartreusin binds cooperatively to poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC). Both the site-exclusion model and the specific site model yield cooperative binding constants of about 5 X 10(5) M-1 and 3 X 10(5) M-1 for the AT and GC polymers, respectively, and the same stoichiometry and intrinsic binding constant for both polymers of 5 nucleotides per binding site and 3.1 X 10(4) M-1. The Scatchard plot for calf thymus DNA is curved in the opposite sense from that of cooperative binding. These binding data did not fit the site-exclusion model with the cooperative binding parameter as a variable nor the specific site, negative-cooperative binding model. The site-exclusion model with a cooperative binding parameter of unity yielded a binding constant of about 4 X 10(4) M-1 and a stoichiometry of about 5 nucleotides per binding site. The same model for transfer and ribosomal RNA yielded binding constants of 5 X 10(3) M-1 and 7 X 10(3) M-1 and stoichiometries of about 13 and 6 nucleotides per binding site, respectively.

Antibiotics, Antineoplastic↗
PubMed1978

Size and secondary structure of avian myeloblastosis virus associated ribosomal RNA: comparison with cellular and precursor ribosomal RNA.

Ribosomal RNA isolated from ribosomes present inside avian myeloblastosis virus (AMV) was characterized by electron microscopy using the formamide-urea spreading technique. The molecular weight and the secondary structures were compared with those of r-RNA and precursor r-NA isolated from host cells, the leukemic myeloblasts. The molecular weight of viral r-RNA (1.62 +/- 0.18 X 10(6) and 0.69 +/- 0.10 X 10(6)) and the molecular weight of cellular r-RNA (1.63 +/- 0.18 X 10(6) and 0.67 +/- 0.09 X 10(6)), the latter obtained from avian myeloblasts, were found to be identical and comparable with the molecular weight of chicken liver r-RNA. Likewise, the secondary structures of viral r-RNA were identical to those of cellular r-RNA. The postulated possible precursor character of viral r-RNA was excluded, since the molecules of viral r-RNA do not show any similarity to those of precursor r-RNA. Previously observed differences in behavior of viral and cellular (myeloblastic) r-RNA in sedimentation and electrophoretic mobility are discussed.

Animals↗
PubMed1993

Small nuclear RNAs in messenger RNA and ribosomal RNA processing.

Ribonucleoproteins (RNPs) play essential roles in many aspects of gene expression. Two families of nuclear RNPs are involved in the processing of primary transcripts made by RNA polymerases I and II (pol I and II), two of the three polymerases present in the nuclei of eukaryotic cells. Ribosomal RNA precursor transcription by pol I, subsequent processing of the precursor, and the initial steps of ribosome assembly all take place in the nucleolus. A group of nucleolar RNPs containing small RNAs (small nucleolar RNAs or snoRNAs) are involved in the posttranscriptional nucleolar events of ribosome biosynthesis. Six members of a related family of small nuclear RNAs (snRNAs) are required for the processing of mRNA precursors in the nucleoplasm. Five of these snRNAs (U1, U2, U4-6) participate in the removal of intervening sequences while the sixth (U7) plays an essential role in the 3' processing of a subset of mRNA precursors, the histone pre-mRNAs. This is a review of structural and functional aspects of the U1-U7 snRNAs and of snoRNAs.

Base Sequence↗
PubMed2001

Mapping 2'-O-methyl groups in ribosomal RNA.

Ribosomal RNAs (rRNAs) from all sources contain modified nucleosides, whose numbers range from a few in mitochondrial rRNA to more than 200 in the complete rRNAs of some higher eukaryotes. In eukaryotic rRNA the great majority of modified nucleosides are 2'-O-methylated nucleosides or pseudouridines. The locations of most of the 2'-O-methylated nucleosides in rRNA from some representative eukaryotes are known from studies whose aim was full characterization of rRNA methylation. More recently, and particularly in connection with the discovery of methylation guide RNAs, it is often required to check for the presence or absence of 2'-O-methyl nucleosides at specified locations within rRNA. Three methods that can be applied for such "local" objectives are reviewed. Two of the methods are based on primer extension by reverse transcriptase. They exploit, respectively, a tendency of 2'-O-methyl groups to impede reverse transcriptase at low dNTP concentrations, or the resistance of phosphodiester bonds adjacent to 2'-O-methyl groups to alkaline hydrolysis. Examples of these methods are summarized. Although the two methods are relatively straightforward, they suffer from various experimental limitations, as discussed. The third method is technically more sophisticated but is capable of overcoming the limitations of the first two methods. It is based on the resistance of a target 2'-O-methylated site to cleavage by RNase H when the site is hybridized to an appropriate chimeric oligonucleotide. An overview of the approaches and methods now available for the complete mapping of 2'-O-methyl groups in rRNA is presented.

Binding Sites↗