Showing papers on "Ribosomal protein published in 1996"

Cold shock stress-induced proteins in Bacillus subtilis.

Abstract: Bacteria respond to a decrease in temperature with the induction of proteins that are classified as cold-induced proteins (CIPs). Using two-dimensional gel electrophoresis, we analyzed the cold shock response in Bacillus subtilis. After a shift from 37 to 15 degrees C the synthesis of a majority of proteins was repressed; in contrast, 37 proteins were synthesized at rates higher than preshift rates. One hour after cold shock, the induction of CIPs decreased, and after 2 h, general protein synthesis resumed. The identified main CIPs were excised from two-dimensional gels and were subjected to microsequencing. Three small acidic proteins that showed the highest relative induction after cold shock were highly homologous and belonged to a protein family of which one member, the major cold shock protein, CspB, has previously been characterized. Two-dimensional gel analyses of a cspB null mutant revealed that CspB affects the level of induction of several CIPs. Other identified CIPs function at various levels of cellular physiology, such as chemotaxis (CheY), sugar uptake (Hpr), translation (ribosomal proteins S6 and L7/L12), protein folding (PPiB), and general metabolism (CysK, Ilvc, Gap, and triosephosphate isomerase).

Specific sequences in the fragile X syndrome protein FMR1 and the FXR proteins mediate their binding to 60S ribosomal subunits and the interactions among them.

Abstract: Fragile X syndrome, the most common form of hereditary mental retardation, usually results from lack of expression of the FMR1 gene. The FMR1 protein is a cytoplasmic RNA-binding protein. The RNA-binding activity of FMR1 is an essential feature of FMR1, as fragile X syndrome can also result from the expression of mutant FMR1 protein that is impaired in RNA binding. Recently, we described two novel cytoplasmic proteins, FXR1 and FXR2, which are both very similar in amino acid sequence to FMR1 and which also interact strongly with FMR1 and with each other. To understand the function of FMR1 and the FXR proteins, we carried out cell fractionation and sedimentation experiments with monoclonal antibodies to these proteins to characterize the complexes they form. Here, we report that the FMR1 and FXR proteins are associated with ribosomes, predominantly with 60S large ribosomal subunits. The FXR proteins are associated with 60S ribosomal subunits even in cells that lack FMR1 and that are derived from a fragile X syndrome patient, indicating that FMR1 is not required for this association. We delineated the regions of FMR1 that mediate its binding to 60S ribosomal subunits and the interactions among the FMR1-FXR family members. Both regions contain sequences predicted to have a high propensity to form coiled coil interactions, and the sequences are highly evolutionarily conserved in this protein family. The association of the FMR1, FXR1, and FXR2 proteins with ribosomes suggests they have functions in translation or mRNA stability.

The fragile X mental retardation protein is associated with ribosomes

Abstract: Fragile X mental retardation syndrome is one of the most common human genetic diseases. Patients carry a methylated expansion of a CGG repeat resulting in the silencing of the FMR1 gene1–5 that codes for a heterogeneous set of proteins (FMRP)6–9. FMRP is abundant in neurons and is also widely expressed, albeit at different levels, in various human and mouse tissues. FMRP is cytoplasmic6,7 and contains two conserved RNA-binding domains, suggesting a possible involvement in RNA metabolism10–12. However, its function remains unknown. To understand the possible role(s) of FMRP in cellular processes, we investigated its association with cellular structures. We observed that FMRP cosediments with polyribosomes after ultracentrifugation in sucrose density gradients. Following the dissociation of ribosomes into their components, we found that FMRP is associated with the ribosomal 60S subunit and possesses the characteristics of a nonintegral ribosomal protein. Immunofluorescence studies reveal a tight colocalisation of FMRP with cytoplasmic ribosomes in NIH 3T3 and HeLa cells and in primary cultures of neurons, confirming our biochemical observations. We propose that fragile X mental retardation might result from defects in the translational machinery due to the absence of FMRP.

RbfA, a 30S ribosomal binding factor, is a cold‐shock protein whose absence triggers the cold‐shock response

Abstract: The cold-shock response, characterized by a specific pattern of gene expression, is induced upon a downshift in temperature and in the presence of inhibitors of ribosomal function. Here, we demonstrate that RbfA of Escherichia coli, considered to be involved in ribosomal maturation and/or initiation of translation, is a cold-shock protein. Shifting the rbfA mutant to a lower temperature resulted in a constitutive induction of the cold-shock response accompanied by slower growth at low temperatures, while shifting the rbfA mutant that overproduces wild-type RbfA resulted in an increase in total protein synthesis accompanied by faster growth adaptation to the lower temperature. Furthermore, the cold-shock response was also constitutively induced in a cold-sensitive 16S rRNA mutant at low temperatures. Accompanying the transient induction of the cold-shock response, we also report that shifting E. coli from 37 degrees C to 15 degrees C resulted in a temporary inhibition of initiation of translation, as evidenced by the transient decrease in polysomes accompanied by the transient increase in 70S monosomes. The accumulative data indicate that the inducing signal for the cold-unadapted non-translatable ribosomes which are converted to cold-adapted translatable ribosomes by the association of cold-shock proteins such as RbfA. Therefore, the expression of the cold-shock response, and thus cellular adaptation to low temperature, is regulated at the level of translation. The data also indicate that cold-shock proteins can be translated by ribosomes under conditions that are not translatable for most mRNAs.

Genomic Diversity and Differentiation among Phytoplasma Strains in 16S rRNA Groups I (Aster Yellows and Related Phytoplasmas) and III (X-Disease and Related Phytoplasmas)

Abstract: Conserved gene sequences, including 16S rRNA and ribosomal protein gene sequences, were used to evaluate genetic variations in phytoplasma strains belonging to 16S rRNA groups I (aster yellows and related phytoplasmas) and III (X-disease and related phytoplasmas). We used PCR to amplify the sequences of the 16S ribosomal DNA and a segment of the ribosomal protein gene operon (encoding the 3' region of rps19, all of rpl22, and rps3) from diverse phytoplasma group I and III strains. Additional chromosomal gene sequences of group I strains were also amplified. The PCR products amplified from members of each group of phytoplasmas were compared by performing restriction fragment length polymorphism (RFLP) analyses. On the basis of the RFLP patterns observed and similarity coefficients derived from combined RFLP analyses, the phytoplasma strains belonging to groups I and III were placed in distinct 16S rRNA, ribosomal protein, and 16S rRNA-ribosomal protein subgroups. Analyses of two or more conserved gene sequences revealed that members of the two groups were more diverse than previously thought. Subgroup differentiation on the basis of our combined analyses of 16S rRNA and ribosomal protein gene sequences seemed to adequately reflect the levels of chromosomal homology determined by DNA-DNA hybridization assays. On the basis of unique RFLP profiles, we identified new, previously unclassified group I phytoplasma strains, including the organisms that are associated with Ipomoea obscura witches'-broom [subgroup 16SrI-F(rr-rp)], maize bushy stunt [subgroup 16SrI-I(rr-rp)], and Mexican periwinkle virescence [subgroup 16SrI-J(rr-rp)], and new, previously unclassified group III phytoplasma strains, including the organism that is associated with pecan bunch [subgroup 16SrIII-H(rr-rp)]. On the basis of the results of our analyses of 16S rRNA and ribosomal protein conserved gene sequences, we recognized 9 group I subgroups and eight group III subgroups. We propose that phytoplasma strains belonging to each group I and III subgroup should be distinguished taxonomically at a level equivalent to the subspecies level.

A transposon insertion in the Arabidopsis SSR16 gene causes an embryo‐defective lethal mutation

Abstract: The SSR16 gene of Arabidopsis has been identified as a gene encoding a ribosomal protein S16 homolog through analysis of a transposon insertion mutation. The insertion mutation is lethal, arresting embryonic development at approximately the transition from the globular to the heart stage of embryonic development. Co-segregation of the mutant phenotype with the transposon-borne drug-resistance marker and loss of the inserted transposon concomitant with phenotypic reversion provided evidence that the transposon had caused the mutation. Sequences flanking the insertion site were amplified from DNA of viable heterozygotes by thermal asymmetric interlaced (TAIL) PCR. The amplified fragment flanking the 3' end of the inserted element was sequenced and found to be identical to an Arabidopsis expressed sequence tag (EST). The EST, in turn, contained a coding sequence homologous to the ribosomal protein S16 (RPS16) of bacteria such as Escherichia coli, Bacillus subtilis and Salmonella typhimurium, as well as Neurospora crassa mitochondria and higher plant plastids. Thus the gene identified by the embryo-defective lethal insertion mutation encodes an RPS16 homolog and has been designated the SSR16 gene.

13 Translational Control of Ribosomal Protein mRNAs in Eukaryotes

Abstract: Many complex processes that occur during normal growth, differentiation, development, or malignant transformation in eukaryotes involve regulation of ribosome biosynthesis. This organelle contains four species of ribosomal RNA and more than 70 different ribosomal protein molecules, all of which appear in equimolar amounts (for review, see Wool et al., this volume). Thus, to maintain the proper stoichiometry of the ribosomal components at varying cellular growth rates, there must be regulatory mechanisms to ensure both coordinate synthesis of all ribosomal components and modulated formation of ribosomes. The equimolar accumulation of ribosomal proteins is maintained by coordinate regulation at various levels of gene expression (Meyuhas et al. 1987; Aloni et al. 1992 and references therein). It seems, however, that under many physiological conditions, control of ribosomal protein gene expression at the translational level is the most prevalent regulatory mechanism operating in both eukaryotes and prokaryotes. Interestingly, the synthesis of ribosomal proteins in yeast is generally not regulated at the translational level (Tsay et al. 1988), with the exception of a single documented case (Dabeva and Warner 1993). This chapter focuses on the translational control of messenger RNAs encoding both ribosomal proteins and several related proteins during development, with fluctuations in growth rate and upon hormonal stimulation, as studied in various eukaryotic organisms and cell lines. Alterations in the translational efficiency of these mRNAs appear to be of a selective nature, to be coregulated with the accumulation of rRNA, and to involve the 5′-untranslated region (5′ UTR) as the cis -regulatory element. GROWTH-DEPENDENT TRANSLATIONAL...

A microsomal ATP-binding protein involved in efficient protein transport into the mammalian endoplasmic reticulum.

Abstract: Protein transport into the mammalian endoplasmic reticulum depends on nucleoside triphosphates. Photoaffinity labelling of microsomes with azido-ATP prevents protein transport at the level of association of precursor proteins with the components of the transport machinery, Sec61 alpha and TRAM proteins. The same phenotype of inactivation was observed after depleting a microsomal detergent extract of ATP-binding proteins by passage through ATP-agarose and subsequent reconstitution of the pass-through into proteoliposomes, Transport was restored by co-reconstitution of the ATP eluate, This eluate showed eight distinct bands in SDS gels. We identified five lumenal proteins (Grp170, Grp94, BiP/Grp78, calreticulin and protein disulfide isomerase), one membrane protein (ribophorin I) and two ribosomal proteins (L4 and L5), In addition to BiP (Grp78), Grp170 was most efficiently retained on ATP-agarose. Purified BiP did not stimulate transport activity, Sequence analysis revealed a striking similarity of Grp170 and the yeast microsomal protein Lhs1p which was recently shown to be involved in protein transport into yeast microsomes. We suggest that Grp170 mediates efficient insertion of polypeptides into the microsomal membrane at the expense of nucleoside triphosphates.

The Large Ribosomal Subunit Stalk as a Regulatory Element of the Eukaryotic Translational Machinery

Abstract: Publisher Summary This chapter reviews the structural and functional properties of different eukaryotic stalk components, focusing on the Saccharomyces cerevisiae system for which an extensive analysis has been carried out. One of the most characteristic structural features of the large ribosomal subunit is the stalk—a highly flexible lateral protuberance that, in bacterial particles, is formed by a pentameric protein complex of two dimers of proteins, L7 and L12, and one copy of protein L10. Proteins L7 and L12—the N-terminal acetylated and nonacetylated forms of a unique, strongly acidic polypeptide—form in solution stable dimers that have been proposed to have an elongated structure with a globular carboxyl domain and a rigid amino end joined by a flexible “hinge.” The corresponding components of the stalk and the GTPase domain in eukaryotic ribosomes have been identified and characterized. They show characteristics that resemble closely those of their bacterial counterparts, and it is generally accepted that they must play an analogous functional role in the basic protein synthesis machinery, facilitating also the interaction of elongation factors.

Mechanism of action of streptogramins and macrolides.

Abstract: Protein synthesis is catalysed by ribosomes and cytoplasmic factors. Bacterial ribosomes (70S) are made up of 2 subunits (50S and 30S) containing ribosomal RNA (rRNA) and ribosomal proteins: the 30S binds messenger RNA and begins the ribosomal cycle (initiation), whereas 50S binds transfer RNA (tRNA) derivatives and controls elongation. The key reaction, peptide bond formation, is promoted by the catalytic centre of 50S (the peptidyl transferase centre), and the growing peptide chain (peptidyl-tRNA) attached at the donor P site undergoes peptide linkage with an aminoacyl-tRNA at the acceptor A site. This reaction is inhibited by several antibiotics, the best known being chloramphenicol, and the macrolide-lincosamide-streptogramin (MLS) group. These inhibitors have a reversible action, except for streptogramins that are composed of A and B components, which are bacteriostatic alone, but bactericidal when combined. The peptidyl transferase centre has been identified at the 50S surface, and the binding sites of inhibitors have been mapped within this domain: some of these sites overlap (e.g. those of macrolides, and type B streptogramins, which compete for binding to ribosomes). Chloramphenicol blocks the catalytic portion, and A streptogramins the substrate sites of the peptidyl transferase centre. Macrolides and type B streptogramins interfere with the formation of long polypeptides and cause a premature detachment of incomplete peptide chains. The synergism between types A and B streptogramins is due to induction by type A streptogramins of an increased ribosome affinity for type B streptogramins. Microbial resistance to antibiotics mainly involves inactivation of inhibitors and modification of targets (mutations of ribosomal proteins or rRNA genes). Alterations of rRNA bases can induce resistance to a single inhibitor or to a group of antibiotics (e.g. MLSB). The impact of resistance in chemotherapy is less important for streptogramins than for other inhibitors, because the synergistic effect of A and B streptogramins also applies to strains resistant to the MLSB group. It is proposed that mutations and modifications of rRNA bases induce conformational ribosomal changes that prevent antibiotics binding to the target. Conformational changes are also triggered by type A streptogramins: they are responsible for their synergism with type B streptogramins.

Identification of Genes Differentially Expressed in B16 Murine Melanoma Sublines with Different Metastatic Potentials

Abstract: B16-F10 and B16-BL6 are B16 mouse melanoma sublines that preferentially metastasize to the lung following i.v. and s.c. injections, respectively. To study molecular mechanisms underlying the different metastatic behaviors exhibited by the B16 melanoma sublines, we performed differential hybridization of the genes transcribed in these cells and compared their expression levels. We isolated four genes that were highly expressed in B16-F10 cells but not in B16-BL6 cells: TI-225 (polyubiquitin), TI-229 (pyruvate kinase), TI-241 (LRF-1 homologue), and TI-227 (novel gene). Triosephosphate isomerase, 10-formyltetrahydrofolate dehydrogenase, tyrosinase-related protein 2, cytochrome c oxidase, ATP synthetase α subunit, RNA helicase, and ribosomal protein (L37, J1, acidic phosphoprotein), however, showed higher expression in B16-BL6 cells than in B16-F10 cells. Among these clones, transfection of TI-241 into the low metastatic clone F1 converted the parental cells from low- into high-metastatic cells. TI-241 may regulate the expression of various genes as a transcription factor in the complex process of metastasis.

Incomplete editing of rps12 transcripts results in the synthesis of polymorphic polypeptides in plant mitochondria.

Abstract: C-to-U editing causes specific nucleotide changes in plant mitochondrial nRNAs that are required for the restoration of the evolutionarily conserved amino acid sequence. Transcripts for the ribosomal protein S12 gene (rps12) have six C-to-U editing sites and are highly heterogeneous as a result of incomplete editing. immunological analysis demonstrated that unedited or partially edited transcripts as well as edited mRNAs are translated. The edited rps12 translation products accumulate as ribosomal subunits, but the unedited rps12 translation products are present as unassembled subunits and are not detected in the ribosomes. Thus, gene expression is polymorphic as a result of incomplete C-to-U editing, and aberrant polypeptides are present from the translation of these mRNAs. However, because only the edited translation products accumulate in mitochondrial ribosomes, the overall expression of rps12 is rendered coherent by the selection

Site-Directed Hydroxyl Radical Probing of the rRNA Neighborhood of Ribosomal Protein S5

Abstract: Cysteine residues were introduced into three different positions distributed on the surface of ribosomal protein S5, to serve as targets for derivatization with an Fe(II)-ethylenediaminetetraacetic acid linker. Hydroxyl radicals generated locally from the tethered Fe(II) in intermediate ribonucleoprotein particles or in 30 S ribosomal subunits reconstituted from derivatized S5 caused cleavage of the RNA, resulting in characteristically different cleavage patterns for the three different tethering positions. These findings provide constraints for the three-dimensional folding of 16 S ribosomal RNA (rRNA) and for the orientation of S5 in the 30 S subunit, and they further suggest that antibiotic resistance and accuracy mutations in S5 may involve perturbation of 16 S rRNA.

Drosophila ribosomal protein PO contains apurinic/apyrimidinic endonuclease activity.

Abstract: Drosophila ribosomal protein PO was overexpressed in Escherichia coli to allow for its purification, biochemical characterization and to generate polyclonal antibodies for Western analysis. Biochemical tests were originally performed to see if overexpressed PO contained DNase activity similar to that recently reported for the apurinic/apyrimidinic (AP) lyase activity associated with Drosophila ribosomal protein S3. The overexpressed ribosomal protein was subsequently found to act on AP DNA, producing scissions that were in this case 5' of a baseless site instead of 3', as has been observed for S3. As a means of confirming that the source of AP endonuclease activity was in fact due to PO, glutathione S-transferase (GST) fusions containing a Factor Xa cleavage site between GST and PO were constructed, overexpressed in an E.coli strain defective for the major 5'-acting AP endonucleases and the fusions purified using glutathione-agarose affinity column chromatography. Isolated fractions containing purified GST-PO fusion proteins were subsequently found to have authentic AP endonuclease activity. Moreover, glutathione-agarose was able to deplete AP endonuclease activity from GST-PO fusion protein preparations, whereas the resin was ineffective in lowering DNA repair activity for PO that had been liberated from the fusion construct by Factor Xa cleavage. These results suggested that PO was a multifunctional protein with possible roles in DNA repair beyond its known participation in protein translation. In support of this notion, tests were performed that show that GST-PO, but not GST, was able to rescue an E.coli mutant lacking the major 5'-acting AP endonucleases from sensitivity to an alkylating agent. We furthermore show that GST-PO can be located in both the nucleus and ribosomes. Its nuclear location can be further traced to the nuclear matrix, thus placing PO in a subcellular location where it could act as a DNA repair protein. Other roles beyond DNA repair seem possible, however, since GST-PO also exhibited significant nuclease activity for both single- and double-stranded DNA.

Isolation of cDNAs representing dithiolethione-responsive genes.

Abstract: Dithiolethiones inhibit tumorigenicity elicited by many structurally diverse carcinogens in numerous target tissues. These protective actions are associated with the induction of several carcinogen detoxification enzymes, some of which have only recently been discovered. In order to identify additional novel inducible detoxification response genes, a cDNA library was prepared from liver of rats treated with 1,2-dithiole-3-thione (D3T) and was screened by a differential hybridization method. Complementary DNA clones for several known D3T-inducible genes were isolated, such as epoxide hydrolase, aflatoxin B1-aldehyde reductase, quinone reductase and multiple subunits of glutathione S-transferase. Clones representing genes not previously associated with detoxification were isolated, including those for ferritin heavy and light subunits, ribosomal proteins L18a and S16 and two novel genes, termed dithiolethione-inducible genes (or DIG-1 and DIG-2). Levels of mRNA recognized by each clone were increased from 2- to 31-fold, with maximum induction between 6 and 30 h after treatment with D3T. Except for epoxide hydrolase, the kinetics of induction of each mRNA was coordinate with increased rates of gene transcription. However, based on the time of response to D3T, at least two sets of responsive genes were identified. One set of genes, including glutathione S-transferase Yp, aflatoxin B1-aldehyde reductase, quinone reductase and DIG-1, had low constitutive and highly inducible expression (approximately 20-fold) and the other, including glutathione S-transferase Ya and Yb, epoxide hydrolase, ferritin heavy and light subunits, ribosomal proteins L18a and S16 and DIG-2, had relatively high constitutive and modestly inducible expression (approximately 5-fold). The simplest explanation for this differential expression of D3T-inducible genes is that multiple regulatory mechanisms govern their response. The transcriptional activation of ferritin, ribosomal protein, DIG-1 and DIG-2 genes in conjunction with those of carcinogen detoxification enzymes suggests that they participate in the pleiotropic cellular defense response to dithiolethiones that inhibits chemically produced tumorigenesis.

Crystal structure of the rna binding ribosomal protein l1 from thermus thermophilus

Abstract: L1 has a dual function as a ribosomal protein binding rRNA and as a translational repressor binding mRNA. The crystal structure of L1 from Thermus thermophilus has been determined at 1.85 angstroms resolution. The protein is composed of two domains with the N- and C-termini in domain I. The eight N-terminal residues are very flexible, as the quality of electron density map shows. Proteolysis experiments have shown that the N-terminal tail is accessible and important for 23S rRNA binding. Most of the conserved amino acids are situated at the interface between the two domains. They probably form the specific RNA binding site of L1. Limited non-covalent contacts between the domains indicate an unstable domain interaction in the present conformation. Domain flexibility and RNA binding by induced fit seems plausible.

Synthesis Rates of Cellular Proteins Involved in Translation and Protein Folding Are Strongly Altered in Response to Overproduction of Basic Fibroblast Growth Factor by Recombinant Escherichia coli

Abstract: The cellular response to temperature-induced production of human basic fibroblast growth (bFGF) factor by recombinant E. coli (bacteriophage lambda PRPL promoter/cI857 repressor expression system) was studied by one- and two-dimensional gel electrophoresis. Temperature shift from 30 to 42 degrees C caused the induction of heat-shock protein synthesis and the repression of synthesis of ribosomal proteins and the protein folding catalyst trigger factor. Compared to control cells, carrying the expression vector without structural bFGF gene cells, producing the heterologous protein exhibited a stronger increase in the synthesis rate of heat-shock proteins ClpB (HtpM), DnaK, HtpG, GroEL, GrpE, and IbpB (HtpE) in response to temperature upshift. Unexpectedly, formation of the chaperone heat-shock protein GroES was not detected after temperature shift to 42 degrees C in cells producing bFGF. In addition to amplified heat-shock protein formation, the syntheses of ribosomal proteins and of the protein folding catalyst trigger factor were more severely repressed after temperature upshift in cells producing bFGF. In conclusion, the normal cellular stress response caused by the high inducing temperature was strongly amplified by heterologous protein synthesis. In particular, syntheses of proteins involved in translation and protein folding were affected by the overproduction of the heterologous protein.