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At least 775 records · Page 43Linked to original sources
PubMed1980

Studies on the function of two adjacent N6,N6-dimethyladenosines near the 3' end of 16S ribosomal RNA of Escherichia coli. IV. The effect of the methylgroups on ribosomal subunit interaction.

The effect of the presence or absence of the methylgroups of the m2(6)Am2(6)A sequence near the 3' end of 16S rRNA of Escherichia coli on the interaction of the ribosomal subunits has been studied, using wild-type (methylated) and mutant (unmethylated) ribosomes. Subunit exchange experiments and competitive association experiments show a strong preference of the 50S subunit for association with methylated 30S subunits. The results indicate that the equilibrium constant of the reaction 70S in equilibrium with 30S + 50S is dependent on the methylgroups; mutant 30S.50S couples are less stable than wild-type 30S.50S couples. It is postulated that the methylgroups also stimulate the interaction between 30S subunits and initiation factor IF-3.

Adenosine↗
PubMed1981

Secondary structure of the large subunit ribosomal RNA from Escherichia coli, Zea mays chloroplast, and human and mouse mitochondrial ribosomes.

Short base-paired RNA fragments, and fragments containing intra-RNA cross-links, were isolated from E. coli 23S rRNA or 50S ribosomal subunits by two-dimensional gel electrophoresis. The interactions thus found were used as a first basis for constructing a secondary structure model of the 23S rRNA. Sequence comparison with the 23S rDNA from Z. mays chloroplasts, as well as with the 16S (large subunit) rDNA from human and mouse mitochondria, enabled the experimental model to be improved and extrapolated to give complete secondary structures of all four species. The structures are organized in well-defined domains, with over 450 compensating base changes between the two 23S species. Some ribosomal structural "'switches" were found, one involving 5S rRNA.

Animals↗
PubMed1979

The pools of ribosomal proteins and ribosomal ribonucleic acids during relaxed control of Escherichia coli A19 (Hfr, rel met rns).

The soluble fraction extracted from Escherichia coli A19 (Hfr, rel met rns) during early and late times of phenotypic and genotypic induced relaxed control have been examined for the possible accumulation of ribosomal proteins (r-proteins) and rRNA species during this time of unbalanced macromolecular synthesis. Ribosomal proteins and rRNA species were not found to accumulate within the soluble fraction at any time during this period of relaxed control; even after the typical rRNA accumulation had ceased, r-proteins did not accumulate. It is concluded, from these and related observations, that the r-proteins and rRNA species known to be produced during relaxation must immediately associate to form the unusual ribonucleoprotein particles (e.g. 'relaxed particles' and 'chloramphenicol particles') characteristic of periods of relaxed control. Since r-proteins do not accumulate even when net RNA accumulation halts, it appears that some elements of the normal, basic co-ordination between rRNA and r-protein synthesis/stability persist even during relaxed control.

Centrifugation, Density Gradient↗
PubMed1976

The binding site of Escherichia coli ribosomal protein S4 on 16-S ribosomal RNA from different bacterial species.

Ribosomal protein S4 of Escherichia coli was bound to 16-S ribosomal RNAs from several bacterial species and the complexes digested with pancreatic ribonuclease in an effort to isolate heterologous RNA binding sites for protein S4. 16-S RNAs from Aeromonas punctata, Pseudomonas fluorescens and Vibrio cuneatus each gave rise to protected fragments whose electrophoretic mobility was 7S, i.e. similar to that of the fragment generated from E. coli 16-S RNA using the same conditions. No comparable fragment was obtained from 16-S RNA of either Bacillus subtilis or Bacillus stearothermophilus, if E. coli protein S4 was present prior to digestion. The protected 7-S RNA fragment from A. punctata and the subfragments obtained from it by gel electrophoresis under denaturing conditions were characterized further by fingerprinting and nucleotide sequence analysis. The sequence of many of the T1 ribonuclease oligonucleotides was obtained and compared to those from the E. coli 7-S fragment. This has permitted a tentative identification of the sequences of A. punctata 16-S RNA which are protected by E. coli protein S4, namely, the regions homologous to the E. coli sequence from section M through C''. The fingerprints of the protected 7-S fragments from both P. fluorescens and V. cuneatus were sufficiently different from that of the E. coli 7-S fragment that no conclusions regarding sequence homologies could be drawn.

Aeromonas↗
PubMed1977

RNA sequences in ribonucleoprotein fragments of the complex formed from ribosomal 23-S RNA and ribosomal protein L24 of Escherichia coli.

Upon digestion of the complex formed from the 23-S ribosomal RNA and the 50-S ribosomal protein L24 of Escherichia coli, two fragments resistant to ribonuclease were recovered; these fragments contained RNA sections belonging to the 480 nucleotides at the 5' end of 23-S RNA. By determining the sequence of 70% of this latter region we were able to localise the sections which, in the presence of the protein, are resistant to ribonuclease. Our results suggest that the region encompassing the 480 nucleotides starting at the 9th nucleotide from the 5' end of 23-S RNA has a compact tertiary structure, which is stabilised by protein L24.

Alkaline Phosphatase↗
PubMed1978

The binding site of ribosomal protein L1 from Escherichia coli on the 23-S ribosomal RNA from Bacillus stearothermophilus. A possible base-pairing scheme differing from that proposed for Escherichia coli.

The region of Bacillus stearothermophilus strain NCA 1503 23-S ribosomal RNA protected from T1 ribonuclease digestion by the 50-S ribosomal subunit protein L1 from Escherichia coli has been established. The sequence of 115 nucleotides is compared to the analogous region in E. coli. The similar behaviour of the RNA towards the recognition of protein L1 may be explained in terms of secondary base-pairing, even though there exists almost 40% difference between the primary nucleotide sequences.

Base Sequence↗
PubMed1978

The modification of the peptidyl transferase activity of 50-S ribosomal subunits, LiC1-split proteins and L16 ribosomal protein by ethoxyformic anhydride.

Ethoxyformic anhydride abolishes the peptidyl transferase activity of 50-S ribosomal subunits, LiC1 split proteins and L16. Hydroxylamine treatment results in reactivation. Erythromycin exhibits significant protection with 50-S ribosomal subunits. With LiC1 split proteins and L16 significant protection was exhibited only after reconstitution. The results indicate that the ethoxyformic anhydride is reacting with approximately six histidines in LiC1 split proteins and one in L16. Since L16 has been reported to contain a single histidine, the results presented indicate the involvement of this histidine in peptidyl transferase activity.

Acyltransferases↗
PubMed1979

50-S subunit from Escherichia coli ribosomes. Isolation of active ribosomal proteins and protein complexes.

A method is described for the isolation of highly purified proteins from the 50-S subunit of Escherichia coli ribosomes. All the proteins from the large subunit could be isolated with the exception of L14, L26, L31 and L34. The isolated proteins are functionally active in reconstituted particles. The method consists of successive NH4Cl/EtOH and LiCl washing steps, which split off distinct groups of proteins from the ribosome. The protein groups are further separated by a combination of gel filtration (Sephadex G-100) and ion-exchange chromatography (carboxymethylcellulose) in the presence of 6 M urea, at neutral pH and 4 degrees C. The purity of the proteins was analyzed by two-dimensional gel electrophoresis. In addition, ten protein complexes were isolated and identified.

Chromatography, Gel↗
PubMed1980

30-S ribosomal subunit proteins of an Escherichia coli mutant in which assembly of the small ribosomal subunit is temperature-sensitive.

Escherichia coli 219ts2, a temperature-sensitive streptomycin-independent revertant of the streptomycin-dependent strain E. coli 209 is defective in 30-S ribosomal subunit assembly at 42 degrees C. Total 30-S ribosomal subunit proteins of this strain contain two additional components one of which (alpha) migrates below and the other (beta) to the right of protein S7 in two-dimensional polyacrylamide gel analyses carried out according to Kaltschmidt and Wittmann. These two components are also resolved from normal 30-S subunit proteins by chromatography on phosphocellulose. Tryptic fingerprinting of proteins alpha and beta identifies alpha as protein S7B, the form of S7 found in B strains of E. coli and beta as a mutant form of protein S4 produced by deletion of about 20 amino acids from the COOH terminus of the wild-type protein.

Amino Acids↗
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

Escherichia coli ribosomal protein L10 is rapidly degraded when synthesized in excess of ribosomal protein L7/L12.

In Escherichia coli the genes encoding ribosomal proteins L10 and L7/12, rplJ and rplL, respectively, are cotranscribed and subject to translational coupling. Synthesis of both proteins is coordinately regulated at the translational level by binding of L10 or a complex of L10 and L7/L12 to a single target in the mRNA leader region upstream of rplJ. Unexpectedly, small deletions that inactivated the ribosome-binding site of the rplL gene carried on multicopy plasmids exerted a negative effect on expression of the upstream rplJ gene. This effect could be overcome by overproduction of L7/L12 in trans from another plasmid. This apparent stimulation resulted from stabilization of the overproduced L10 protein by L7/L12, presumably because free L10, in contrast to L10 complexed with L7/L12, is subject to rapid proteolytic decay. The contribution of this decay mechanism to the regulation of the rplJL operon is evaluated.

Bacterial Proteins↗
PubMed2004

Ribosomal protein L23 activates p53 by inhibiting MDM2 function in response to ribosomal perturbation but not to translation inhibition.

The p53-MDM2 feedback loop is vital for cell growth control and is subjected to multiple regulations in response to various stress signals. Here we report another regulator of this loop. Using an immunoaffinity method, we purified an MDM2-associated protein complex that contains the ribosomal protein L23. L23 interacted with MDM2, forming a complex independent of the 80S ribosome and polysome. The interaction of L23 with MDM2 was enhanced by treatment with actinomycin D but not by gamma-irradiation, leading to p53 activation. This activation was inhibited by small interfering RNA against L23. Ectopic expression of L23 reduced MDM2-mediated p53 ubiquitination and also induced p53 activity and G(1) arrest in p53-proficient U2OS cells but not in p53-deficient Saos-2 cells. These results reveal that L23 is another regulator of the p53-MDM2 feedback regulation.

Amino Acid Sequence↗
PubMed1985

Posttranscriptional regulation and assembly into ribosomes of a Saccharomyces cerevisiae ribosomal protein-beta-galactosidase fusion.

To study the regulation of ribosomal protein genes, we constructed a 'lacZ fusion of the Saccharomyces cerevisiae RP51A gene, containing the first 64 codons of RP51A. In a strain lacking an intact RP51A gene (cells are viable due to the presence of an active RP51B gene), beta-galactosidase activity is 10-fold greater than in a strain containing RP51A. RP51A-lacZ mRNA levels are equal in the two strains, indicating that regulation is posttranscriptional. In the absence of the RP51A gene, the fusion protein is predominantly cytoplasmic and associated with polysomes, whereas in the presence of RP51A, the fusion protein is predominantly nuclear, and none is associated with polysomes. Deletions were made in the RP51A-coding portion of the fusion gene. The most extensively deleted gene, containing only the first seven RP51A codons fused to lacZ, produced a high level of beta-galactosidase activity in both the presence and the absence of the RP51A gene. In both cases, little or none of this shorter fusion protein was found associated with polysomes. Thus, a regulatory site (or sites) lies in the protein-coding region of RP51A. We suggest that posttranscriptional regulation of the rp51 fusion protein is related to assembly of the protein into ribosomes.

Escherichia coli↗
PubMed1988

Gene dosage alteration of L2 ribosomal protein genes in Saccharomyces cerevisiae: effects on ribosome synthesis.

In Saccharomyces cerevisiae, the genes coding for the ribosomal protein L2 are present in two copies per haploid genome. The two copies, which encode proteins differing in only a few amino acids, contribute unequally to the L2 mRNA pool: the L2A copy makes 72% of the mRNA, while the L2B copy makes only 28%. Disruption of the L2B gene (delta B strain) did not lead to any phenotypic alteration, whereas the inactivation of the L2A copy (delta A strain) produced a slow-growth phenotype associated with decreased accumulation of 60S subunits and ribosomes. No intergenic compensation occurred at the transcriptional level in the disrupted strains; in fact, delta A strains contained reduced levels of L2 mRNA, whereas delta B strains had almost normal levels. The wild-type phenotype was restored in the delta A strains by transformation with extra copies of the intact L2A or L2B gene. As already shown for other duplicated genes (Kim and Warner, J. Mol. Biol. 165:79-89, 1983; Leeret al., Curr. Genet. 9:273-277, 1985), the difference in expression of the two gene copies could be accounted for via differential transcription activity. Sequence comparison of the rpL2 promoter regions has shown the presence of canonical HOMOL1 boxes which are slightly different in the two genes.

Base Sequence↗
PubMed2001

Comparison of the embryotoxic effects of saporin, agrostin (type 1 ribosome-inactivating proteins) and ricin (a type 2 ribosome-inactivating protein).

The effects of two type I ribosome-inactivating proteins, saporin and agrostin, and the type 2 ribosome-inactivating protein ricin and its constituent A and B chains, on the development of cultured mouse embryos, were investigated. Saporin and agrostin had similar embryotoxicity which was approximately 10(5) times weaker than that of ricin and its A and B chains, and the embryotoxicity of saporin and agrostin increased gradually with the dosage. In contrast, there was an abrupt rise in embryotoxictiy of ricin-A chain and B chain as the dose was raised from 10 to 20 ng x ml(-1). Saporin and agrostin did not affect the heart-beat and otic placode, but at 10 ng x ml(-1) ricin and 40 ng x ml(-1) ricin-A chain and B chain, all of the treated embryos exhibited abnormalities in heart-beat, yolk sac circulation, body axis, optic placode, otic placode, forelimb buds, branchial apparatus and cranial neural tube.

Animals↗
PubMed1979

Binding of mitochondrial ribosomal proteins to a mitochondrial ribosomal precursor RNA containing a 2.3-kilobase intron.

In Neurospora, the gene encoding the mitochondrial large subunit (25 S) rRNA contains an intervening sequence of approximately 2.3 kilobases. We have recently identified two temperature-sensitive, nuclear mutants (289-67 and 299-9) which are defective in splicing of the 25 S RNA. When grown at the nonpermissive temperature (37 degrees C), the mutants show decreased ratios of 25 S/19 S RNA and accumulate a novel 35 S RNA which appears to be a continuous transcript of the 25 S RNA gene, including the intervening sequence. In the present work, mitochondrial ribonucleoprotein particles present in the 50 S subunit peak from wild type and mutant 299-9 were analyzed by equilibrium centrifugation in CsCl gradients containing 25 mM MgCl2. The results show that 35 S RNA can be isolated as part of a ribonucleoprotein particle associated with nearly all of the large subunit ribosomal proteins. However, the particles appear to be less stable in CsCl gradients and more sensitive to nucleolytic degradation than particles derived from mature large subunits. Our results indicate that binding of ribosomal proteins to 35 S RNA could precede removal of the intron, but that removal of the intron may be required to achieve stable protein binding.

Base Sequence↗
PubMed1990

Ribosomal proteins L15 and L16 are mere late assembly proteins of the large ribosomal subunit. Analysis of an Escherichia coli mutant lacking L15.

The (minus L15) character from the Escherichia coli strain AM16.98 was transduced to an RNase-deficient strain in order to enable a reconstitution analysis. The following results were obtained. 1) The strain lacking L15 showed a 2-3-fold prolonged generation time and the 70 S ribosomes a reduced tendency toward dissociation. 2) Active particles could not be reconstituted unless L15 was added. Addition of L15 regained activity, even if L15 was added after the two-step procedure during a third incubation. However, a modification of the standard two-step reconstitution procedure (lowering NH4+ from 400 to 240 mM and the incubation temperature of the second step from 50 to 47 degrees C) yielded 100% active particles in the absence of L15. Active particles could be formed which even lacked L15, L16, and L30. Addition of either L15 or L16 accelerated the formation of active particles in the second step by a factor of five, and both proteins together by a factor of more than 20. 3) The activation energy of the rate-limiting step of the second incubation was surprisingly reduced for about 20 kcal/mol in the absence of L15, although the corresponding rates were two to five times slower. We conclude 1) that L15 and L16 are late assembly proteins which accelerate the formation of active particles during the late assembly but are neither needed for the early assembly nor essential for ribosomal functions; 2) that some routes of the late assembly (e.g. incorporation of L16) are changing their significance depending on the NH4+ concentration and the absence and presence of L15; and 3) that different reactions are rate limiting during the second step incubation in the presence and absence of L15, respectively, and that the corresponding reaction rates exhibit a different temperature dependence.

Chromosome Deletion↗
PubMed1990

Steroid hormone regulation of ribosomal RNA in rat hypothalamus: early detection using in situ hybridization and precursor-product ribosomal DNA probes.

In the female rat, behavioral and endocrine aspects of reproduction are controlled, in part, by the action of the steroid hormone estradiol on several regions of the brain, including the ventrolateral portion of the ventromedial hypothalamus (VL-VMN) and the arcuate nucleus of the hypothalamus (ARC). Quantitative assessment of the effects of estradiol on the regulation of ribosomal RNA in rat hypothalamus was accomplished in this study by tandem in situ hybridization experiments with 2 ribosomal DNA probes specific to the initial transcript (precursor) or mature, stable (product) rRNA. This novel approach allowed the regulation of RNA processing by steroid hormones to be analyzed in the individual neuron, a particularly important concern in heterogeneous tissue such as the brain. Estradiol was administered subcutaneously to ovariectomized rats for 15 min, 30 min, or 2 hr, or a discontinuous schedule of 2 hr on/7 hr off/2 hr on. Levels of precursor and product rRNA were measured in VL-VMN and ARC neurons using a computerized image-analysis system. Significant increases in the levels of precursor rRNA were observed only in the VL-VMN as early as 30 min after hormone exposure, with a doubling in the amount of precursor rRNA occurring at 2 hr. No changes in product rRNA were observed in either brain region at these early times. These data, in conjunction with our previous findings of increases in product rRNA after longer hormone exposure times, lead us to conclude that rRNA gene transcription is activated in rat hypothalamic neurons within 30 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗