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PubMed1982

Structural organization of rat ribosomal genes restriction endonuclease analysis of genomic and cloned ribosomal DNAs.

Structural organization of the rat ribosomal repeating unit was studied using hybridization of blotted restriction fragments of total rat DNA with alpha-32P-labeled cDNA probe synthesized on the 18S and 28S rRNAs. A detailed restriction endonuclease map was constructed, the 18S and 28S rRNA genes mapped and the sizes of the rat ribosomal repeating units determined. Considerable site heterogeneity of rat rDNA was revealed in both nontranscribed and external transcribed spacers. Recombinant phages containing the whole set of transcribed regions and a considerable part of a nontranscribed spacer of the rDNA were selected from the rat gene library. The restriction maps of the cloned rDNA fragments are in good agreement with the map constructed by Southern's technique, add to this map and support the existence of site heterogeneity in ribosomal repeating units. Proximal to the 3'-end of the 28S rRNA gene an internally repetitive region was found, each repeating unit being equal to approx. 150 bp. The site for transcription initiation was mapped 4.0-4.5 kb upstream from the 5'-end of the 18S rRNA gene. Frequently reiterated interspersed sequences were found in the nontranscribed spacer at approx. 2-3 kb distance from both ends of the transcribed region.

Animals↗
PubMed2003

Assembly of the central domain of the 30S ribosomal subunit: roles for the primary binding ribosomal proteins S15 and S8.

Assembly of the 30S ribosomal subunit occurs in a highly ordered and sequential manner. The ordered addition of ribosomal proteins to the growing ribonucleoprotein particle is initiated by the association of primary binding proteins. These proteins bind specifically and independently to 16S ribosomal RNA (rRNA). Two primary binding proteins, S8 and S15, interact exclusively with the central domain of 16S rRNA. Binding of S15 to the central domain results in a conformational change in the RNA and is followed by the ordered assembly of the S6/S18 dimer, S11 and finally S21 to form the platform of the 30S subunit. In contrast, S8 is not part of this major platform assembly branch. Of the remaining central domain binding proteins, only S21 association is slightly dependent on S8. Thus, although S8 is a primary binding protein that extensively contacts the central domain, its role in assembly of this domain remains unclear. Here, we used directed hydroxyl radical probing from four unique positions on S15 to assess organization of the central domain of 16S rRNA as a consequence of S8 association. Hydroxyl radical probing of Fe(II)-S15/16S rRNA and Fe(II)-S15/S8/16S rRNA ribonucleoprotein particles reveal changes in the 16S rRNA environment of S15 upon addition of S8. These changes occur predominantly in helices 24 and 26 near previously identified S8 binding sites. These S8-dependent conformational changes are consistent with 16S rRNA folding in complete 30S subunits. Thus, while S8 binding is not absolutely required for assembly of the platform, it appears to affect significantly the 16S rRNA environment of S15 by influencing central domain organization.

Binding Sites↗
PubMed2002

Translational feedback regulation of the gene for L35 in Escherichia coli requires binding of ribosomal protein L20 to two sites in its leader mRNA: a possible case of ribosomal RNA-messenger RNA molecular mimicry.

In addition to being a component of the large ribosomal subunit, ribosomal protein L20 of Escherichia coli also acts as a translational repressor. L20 is synthesized from the IF3 operon that contains three cistrons coding for IF3, and ribosomal proteins L35 and L20. L20 directly represses the expression of the gene encoding L35 and the expression of its own gene by translational coupling. All of the cis-acting sequences required for repression by L20, called the operator, are found on an mRNA segment extending from the middle of the IF3 gene to the start of the L35 gene. L20-mediated repression requires a long-range base-pairing interaction between nucleotide residues within the IF3 gene and residues just upstream of the L35 gene. This interaction results in the formation of a pseudoknot. Here we show that L20 causes protection of nucleotide residues in two regions of the operator in vitro. The first region is the pseudoknot itself and the second lies in an irregular stem located upstream of the L35 gene. By primer extension analysis, we show that L20 specifically induces reverse transcriptase stops in both regions. Therefore, these two regions define two L20-binding sites in the operator. Using mutations and deletions of rpml'-'lacZ fusions, we show that both sites are essential for repression in vivo. However L20 can bind to each site independently in vitro. One site is similar to the L20-binding site on 23S rRNA. Here we propose that L20 recognizes its mRNA and its rRNA in similar way.

5' Untranslated Regions↗
PubMed1981

An electron microscopic method for localization of ribosomal proteins during transcription of ribosomal DNA: a method for studying protein assembly.

We describe a method for the localization of ribosomal proteins on electron microscopic spreads of active rRNA genes. The method consists of raising antibodies against Drosophila melanogaster proteins and allowing these antibodies to react with lysates of D. melanogaster egg chambers. The locations of the bound IgGs in the active transcripts are detected with goat anti-rabbit IgGs that have been labeled with electron-dense polymethacrylate spheres. By statistical analysis we can generate a confidence interval for the initial point of protein assembly for a particular protein. The first point of protein assembly for S14 is located near the 5' end of the pre-18S rRNA. In contrast, the first point of protein assembly for L4 is at 0.38 unit from the initiation point (a unit being the length of a ribosomal transcription unit). The binding patterns of S14 and L4 are consistent with the 5' proximal and the 5' distal orientations of the pre-18S and the pre-28S rRNAs. The method described here provides an approach to the elucidation of the assembly of eukaryotic ribosomal proteins in vivo.

Animals↗
PubMed2001

Genes encoding ribosomal proteins Rps0A/B of Saccharomyces cerevisiae interact with TOM1 mutants defective in ribosome synthesis.

The Saccharomyces cerevisiae RPS0A/B genes encode proteins of the 40S ribosomal subunit that are required for the maturation of 18S rRNA. We show here that the RPS0 genes interact genetically with TOM1. TOM1 encodes a member of the hect-domain-containing E3 ubiquitin-protein ligase family that is required for growth at elevated temperatures. Mutant alleles of the RPS0 and TOM1 genes have synergistic effects on cell growth at temperatures permissive for TOM1 mutants. Moreover, the growth arrest of TOM1 mutants at elevated temperatures is partially suppressed by overexpression of RPS0A/B. Strains with mutant alleles of TOM1 are defective in multiple steps in rRNA processing, and interactions between RPS0A/B and TOM1 stem, in part, from their roles in the maturation of ribosomal subunits. Ribosome synthesis is therefore included among the cellular processes governed by members of the hect-domain-containing E3 ubiquitin-protein ligase family.

Alleles↗
PubMed1985

Specific ribosomal RNA recognition by a fragment of E. coli ribosomal protein S4 missing the C-terminal 36 amino acid residues.

We have previously investigated the role of the N-terminal region of ribosomal protein S4 to participate in 30S ribosome assembly and function (1-3). In this report we extend these studies to the two fragments produced by the chemical cleavage of protein S4 at the tryptophan residue 167. We find that the carboxyl terminal fragment (168-203) does not bind 16S RNA nor does it participate in assembly with the other 20 proteins from the 30S ribosome. In contrast, the larger fragment (1-167), does bind 16S RNA specifically. If the S4-fragment (1-167) is used to replace protein S4 in the complete 30S assembly reaction, all 20 of the other 30S proteins are incorporated. We conclude that the carboxyl terminal section of the protein S4 is not directly involved in binding 16S RNA or in the assembly of any of the other 30S proteins.

Amino Acids↗
PubMed1993

Molecular basis of streptomycin resistance in Mycobacterium tuberculosis: alterations of the ribosomal protein S12 gene and point mutations within a functional 16S ribosomal RNA pseudoknot.

Multidrug-resistant strains of Mycobacterium tuberculosis have resulted in several recent outbreaks. Recognition of drug resistance is important both for treatment and to prevent further transmission. Here we use molecular biology techniques to study the basis of streptomycin resistance in single and multidrug-resistant M. tuberculosis. We demonstrate that streptomycin resistance is associated with mutations implicated in ribosomal resistance. The mutations found either lead to amino acid changes in ribosomal protein S12 or alter the primary structure of the 16S rRNA. The 16S rRNA region mutated perturbs a pseudoknot structure in a region which has been linked to ribosomal S12 protein.

Base Sequence↗
PubMed2000

An enzymatic footprinting analysis of the interaction of 40S ribosomal subunits with the internal ribosomal entry site of hepatitis C virus.

Hepatitis C virus translation is initiated on a approximately 330-nucleotide (nt)-long internal ribosomal entry site (IRES) at the 5' end of the genome. In this process, a 43S preinitiation complex (comprising a 40S ribosomal subunit, eukaryotic initiation factor 3 (eIF3), and a ternary [eIF2-GTP-initiator tRNA] complex) binds the IRES in a precise manner so that the initiation codon is placed at the ribosomal P site. This binding step involves specific interactions between the IRES and different components of the 43S complex. The 40S subunit and eIF3 can bind to the IRES independently; previous analyses revealed that eIF3 binds specifically to an apical half of IRES domain III. Nucleotides in the IRES that are involved in the interaction with the 40S subunit were identified by RNase footprinting and mapped to the basal half of domain III and in domain IV. Interaction sites were identified in locations that have been found to be essential for IRES function, including (i) the apical loop residues GGG(266-268) in subdomain IIId and (ii) the pseudoknot. Extensive protection from RNase cleavage also occurred downstream of the pseudoknot in domain IV, flanking both sides of the initiation codon and corresponding in length to that of the mRNA-binding cleft of the 40S subunit. These results indicate that the 40S subunit makes multiple interactions with the IRES and suggest that only nucleotides in domain IV are inserted into the mRNA-binding cleft of the 40S subunit.

Animals↗
PubMed2000

Nitric oxide-dependent ribosomal RNA cleavage is associated with inhibition of ribosomal peptidyl transferase activity in ANA-1 murine macrophages.

NO can regulate specific cellular functions by altering transcriptional programs and protein reactivity. With respect to global cellular processes, NO has also been demonstrated to inhibit total protein synthesis and cell proliferation. The underlying mechanisms are unknown. In a system of ANA-1 murine macrophages, iNOS expression and NO production were induced by exposure to endotoxin (LPS). In selected instances, cells were exposed to an exogenous NO donor, S-nitroso-N-acetylpenicillamine or a substrate inhibitor of NO synthesis. Cellular exposure to NO, from both endogenous and exogenous sources, was associated with a significant time-dependent decrease in total protein synthesis and cell proliferation. Gene transcription was unaltered. In parallel with decreased protein synthesis, cells exhibited a distinctive cleavage pattern of 28S and 18S rRNA that were the result of two distinct cuts in both 28S and 18S rRNA. Total levels of intact 28S rRNA, 18S rRNA, and the composite 60S ribosome were significantly decreased in the setting of cell exposure to NO. Finally, 60S ribosome-associated peptidyl transferase activity, a key enzyme for peptide chain elongation, was also significantly decreased. Our data suggest that NO-mediated cleavage of 28S and 18S rRNA results in decreased 60S ribosome associated peptidyl transferase activity and inhibition of total protein synthesis.

Animals↗
PubMed1976

Proteins of animal ribosomes. XXV. Proteins of rat liver ribosomes protected by polyuridylic acid from methyl acetimidate substitution.

Addition of poly(U) to complexes of 40S and 60S subunits of rat liver ribosomes decreases the substitution of amino groups of 12 proteins of the small ribosomal subunit and of 11 proteins of the large subunit by [14C]-methyl acetimidate. When comparing the results obtained with this amino group specific reagent with the reactivity of the proteins against iodoacetamide it becomes obvious that 4 proteins of the small ribosomal subunit (S12, 18, 19, 24) and 3 proteins of the large one (L20, 22, 25) are partially protected by poly(U) against reaction with both reagents.

Animals↗
PubMedDate not supplied

[The relative content of oocyte and somatic 5S rRNA at different stages of embryonic development as an index of the replacement of maternal ribosomes by ribosomes of the embryo in the loach].

Relative content of the oocyte and somatic 5S rRNA in loach Misgurnus fossilis L. during development was determined electrophoretically. Embryos before hatching contain 70% and swimming larvae no less than 50% of the oocyte 5S rRNA. We assume that the relative content of 5S rRNA fractions reflects the proportion between ribosomes synthesized during oogenesis and those synthesized in embryos and larvae. We calculated using previous data (Timofeeva, Kafiani, 1964) the rates of maternal ribosome decay and ribosome synthesis in the embryo. During organogenesis these rates appear to be 1.17-1.09 x 10(6) and 1.7 x 10(6) molecules/sec per embryo, respectively.

Animals↗
PubMed1981

Phosphorylation of ribosomal proteins as a possible control system for protein synthesis. Binding of Met-tRNAf to 40 S ribosomal subunits.

The evidence that protein S6 of rat liver ribosomes is involved in P-site functions and that this protein is the main target of the small subunit for in vivo phosphorylation suggests that S6 phosphorylation may contribute to the regulation of protein synthesis. Therefore, we have studied the activity of small ribosomal subunits with unphosphorylated and phosphorylated protein S6 in Met-tRNAf binding. The results described in this paper show that at least under in vitro conditions S6 phosphorylation does obviously not influence the activity of small ribosomal subunits for eIF-2 dependent binding of initiator-tRNA.

Animals↗
PubMed1981

Characterization of the binding of rat liver ribosomal proteins L6, L8, L19, S9, and S13 to 5.8 S ribosomal ribonucleic acid.

The interaction of rat liver ribosomal proteins L6, L8, L19, S9, and S13 with 5.8 rRNA was characterized by nitrocellulose membrane filtration. Binding approached saturation with the five proteins; the apparent association constants (K'a), measured at 4 degrees C and 22 degrees C, ranged from 0.2 to 18 X 10(5) M-1. The molar ratio of ribosomal protein and rRNA in the complexes at saturation approximated 1, indicating there is one binding site for each of the five proteins on the nucleic acid. A number of ribosomal proteins, including some previously suspected from affinity chromatography of associating weakly, did not form a complex with 5.8 S rRNA.

Animals↗
PubMed2002

A ribosome-free extract from cultured cells improves recovery of polysomes from the mosquito fat body: analysis of vitellogenin and ribosomal protein rpL34 gene expression.

We have examined the association of ribosomal protein rpL34 mRNA with polysomes in Aedes albopictus C7-10 cells in culture using a simple, two-step sucrose gradient. In growing cells, 40-50% of the ribosomes were engaged on polysomes. This proportion could be increased to 80% when metabolism was stimulated by refeeding the cells with fresh medium. Conversely, ribosomes shifted off polysomes when cells were starved with phosphate-buffered saline or cell lysates were treated with puromycin. When similar approaches were used with fat body from blood-fed female Aedes aegypti mosquitoes, we were unable to obtain the polysome fraction that contained vitellogenin mRNA, which is abundantly translated after a blood meal. Addition of post-mitochondrial supernatant from fat body to polysomes from cultured cells shifted the polysome profile towards smaller polysomes and monosomes, in a dose-dependent fashion. Disruption of fat body tissue in a post-ribosomal supernatant from refed cells improved the recovery of polysomes, demonstrating both the engagement of vitellogenin mRNA on polysomes and the mobilization of rpL34 from messenger-ribonuceloprotein particles onto polysomes in blood-fed mosquitoes. These observations suggested that ribonucleases remain active when polysomes are prepared from mosquito fat body, and that cell culture supernatant contains a ribonuclease inhibitor.

Journal Article↗
PubMed1975

Studies on Pea Ribosomal Proteins: Conformational and Biological Activity Changes of Ribosomal Subunits Derived by NH(4)Cl Dissociation.

Ribosomal subunits prepared by NH(4)Cl dissociation (0.5 m) of the monomeric ribosomes were much less active in in vitro protein synthesis than those prepared by KCl dissociation. The decrease in activity correlated with a detachment of some proteins (L(2) and L(9) as shown by gel electrophoresis) within the 60S ribosomal subunits. Subunits prepared with 0.3 m NH(4)Cl retained L(2) and L(9), but the activity remained low. Incubation of these 60S subunits in TKM buffer (50 mm tris [pH 7.5], 20 mm KCl, and 5 mm MgCl(2)) for 20 min at 37 C restored the activity almost to the level of those obtained by KCl dissociation. Treatment of the 0.3 m NH(4)Cl-derived 60S subunits with a protein reagent, Procion brilliant blue, prior to extraction of the ribosomal proteins resulted in the loss of L(2) and L(9), showing that these proteins were made accessible for dye binding. These observations suggest that a considerable degree of unfolding of the 60S subunit occurs at 0.3 m NH(4)Cl (this apparently leads to a preferential detachment of L(2) and L(9) at 0.5 m NH(4)Cl) and that the activity of the purified subunits depends not only on the presence of L(2) and L(9) but also on the organization of these proteins within the 60S subunits.

Journal Article↗
PubMed2004

Myosin Va and kinesin II motor proteins are concentrated in ribosomal domains (periaxoplasmic ribosomal plaques) of myelinated axons.

Periaxoplasmic ribosomal plaques (PARPs) are discrete ribosome-containing domains distributed intermittently along the periphery of axoplasm in myelinated fibers. Thus, they are structural formations in which translational machinery is spatially organized to serve as centers of protein synthesis for local metabolic requirements and perhaps repair as well. Because of evidence that RNA is transported to putative PARP domains, involving both microtubule- and actin-based mechanisms, it was of interest to investigate whether cytoskeletal motor proteins exhibit a nonrandom localization within PARP domains. Axoplasm, from large Mauthner fibers and rat or rabbit spinal ventral nerve root fibers, removed from the myelin sheath in the form of an "axoplasmic whole-mount" was used for this analysis. PARP domains were identified either by specific immunofluorescence of rRNA, ribosomal P antigen, or by nonspecific RNA fluorescence using RNA binding dyes YOYO-1 or POPO-1. A polyclonal antibody (pAb) against the motor domain of myosin Va showed prominent nonrandom immunofluorescence labeling in PARP domains. Similarly, monoclonal antibodies (mAb) against kinesin KIF3A and a pan-specific antikinesin (mAb IBII) also showed a preponderant immunofluorescence in PARP domains. On the other hand, H2, a mAb antikinesin KIF5A, exhibited only random immunofluorescence labeling in axoplasm, as was also the case with pAb antidynein heavy chain immunofluorescence. Several possible explanations for these findings are considered, primary among which is targeted trafficking of translational machinery that results in local accumulation of motor proteins. Additional possibilities are trafficking functions intrinsic to the domain, and/or functions that govern dynamic organizational properties of PARPs.

Animals↗
PubMed1981

Methylation of ribosomal proteins during ribosome assembly in Escherichia coli.

In Escherichia coli, a number of ribosomal proteins are methylated. The time of methylation of L7 and L11 during ribosome assembly was studied. It was observed that the methylation of L7 could occur in the free protein stage. Both the 32S and 40S ribonucleoprotein intermediates also contained methylated L7 although the extent of methylation in these particles was not as high as in the free L7, the 45S or the 50S particles. Free L11 could also be partially methylated but the bulk of methylation of this protein was found in the 45S and the 50S particles. It was previously reported that the methylation of L7 is inversely proportional to the growth temperature (Chang 1978), we now show that once L7 is methylated at 25 degree, the methyl group is stable when the culture is shifted to 37 degree C. However, a partial turnover of the methyl group of L7 is observed when the methylated ribosome is chased at 25 degree C. On the other hand, the methyl groups of L11 appear to be stable at either 25 degree C or 37 degree C. We also observe that the extent of methylation of both L7 and L11 stays nearly constant during the cell growth cycle from early log to stationary phase.

Escherichia coli↗
PubMed1992

Extrachromosomal ribosomal DNA of Didymium iridis: sequence analysis of the large subunit ribosomal RNA gene and sub-telomeric region.

The ribosomal DNA of the myxomycete Didymium iridis is organized as extrachromosomal linear molecules of about 20 kb, containing only one transcription unit of the ribosomal RNA genes. We have determined the sequence of the large subunit ribosomal RNA (LSU rRNA) gene as well as the sub-telomeric and telomeric regions. The LSU rRNA gene was found to encode a 3857 nucleotide-long LSU rRNA, interrupted by a transcribed spacer and two group I introns. A complete secondary structure model of D. iridis LSU rRNA has been constructed. The compact sub-telomeric region of D. iridis rDNA was found to contain several directly repeated sequence elements that include the simple telomere motif TTAGGG. Based on pairwise comparisons of LSU rRNA sequences, the time of divergence between the two myxomycete genera Didymium and Physarum was estimated.

Animals↗