Showing papers on "Ribosomal protein published in 1985"

Lupus autoantibodies target ribosomal P proteins.

Abstract: All nine SLE (systemic lupus erythematosus) sera with antiribosomal antibody activity targeted the same three ribosomal protein antigens, of molecular masses 38 and 17/19 kD when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. One serum reacted with an additional protein of approximately kD. Ribosomal subunit fractionation by composite gel electrophoresis and sucrose density ultracentrifugation showed that these proteins were part of the large subunit. Isoelectric focusing in agarose, and two-dimensional polyacrylamide gel electrophoresis revealed that the antigens had pI between 4.5 and 6.5, but that the 17/19 kD antigens were more acidic than the 38 kD antigen. Similarities in the molecular masses, charges, as well as the presence of highly conserved crossreactive epitopes, failure to bind to carboxymethylcellulose at pH 4.2, and extractability of the 17/19 kD proteins by 400 mM NH4Cl-ethanol at 0 degrees C indicated that these antigens were analogous to the proteins P0 (38 kD) and P1/P2 (17/19 kD) described previously (25, 36). Co-identity was confirmed using reference antibodies and antigen. Although antibodies to these proteins were only found in 5-10% of more than 50 sera screened by radioimmunoassay or Western blotting, the selective production of antibodies to epitopes on three (out of a total of more than 80) ribosomal proteins may provide further clues to autoantibody induction of SLE.

A Drosophila Minute gene encodes a ribosomal protein.

Abstract: Minute genes have long constituted a special problem in Drosophila genetics. For at least 50-60 different genes scattered throughout the genome, dominant mutations and/or deficiencies have been recognized which result in a common phenotype consisting of short thin bristles, slow development, reduced viability, rough eyes, small body size and etched tergites. Schultz proposed that the Minute loci encode similar but separate functions involved in growth and division common to all cells. Atwood and Ritossa suggested that Minute loci encode components of the protein synthetic machinery, specifically the transfer RNA genes; this now seems unlikely on grounds of both mapping and mutability studies. More recently, we and others suggested that the Minute loci are ribosomal protein genes. We report here that transformation with a cloned 3.3-kilobase (kb) region containing the gene encoding the large subunit ribosomal protein 49 (rp49) suppresses the dominant phenotypes of Minute (3)99D, a previously undescribed Minute associated with a chromosomal deficiency of the 99D interval. This activity is specific to the 99D Minute as it does not suppress other Minute loci elsewhere in the genome. This result provides direct evidence that the Minute locus at the 99D interval encodes the ribosomal protein 49.

Saccharomyces cerevisiae coordinates accumulation of yeast ribosomal proteins by modulating mRNA splicing, translational initiation, and protein turnover.

Abstract: The rate of accumulation of each ribosomal protein is carefully regulated by the yeast cell to provide the equimolar ratio necessary for the assembly of the ribosome. The mechanisms responsible for this regulation have been examined by introducing into the yeast cell extra copies of seven individual ribosomal protein genes carried on autonomously replicating plasmids. In each case studied the plasmid-borne gene was transcribed to the same degree as the genomic gene. Nevertheless, the cell maintained a balanced accumulation of ribosomal proteins, using a variety of methods other than transcription. (i) Several ribosomal proteins were synthesized in substantial excess. However, the excess ribosomal protein was rapidly degraded. (ii) The excess mRNA for two of the ribosomal protein genes was translated inefficiently. We provide evidence that this was due to inefficient initiation of translation. (iii) The transcripts derived from two of the ribosomal protein genes were spliced inefficiently, leading to an accumulation of precursor RNA. We present a model which proposes the autogenous regulation of mRNA splicing as a eucaryotic parallel of the autogenous regulation of mRNA translation in procaryotes. Finally, the accumulation of each ribosomal protein was regulated independently. In no instance did the presence of excess copies of the gene for one ribosomal protein affect the synthesis of another ribosomal protein.

Amino acid sequence of the alpha subunit of transducin deduced from the cDNA sequence

Abstract: Transducin, a GTP-binding protein involved in phototransduction in the vertebrate retina, belongs to a family of homologous coupling proteins that also includes Gs and Gi, the regulatory proteins of adenylate cyclase. Here we report the cDNA sequence and deduced amino acid sequence of transducin's alpha subunit (T alpha). The cDNA was isolated, by screening with an antibody probe, from a bovine retinal cDNA library in the expression vector lambda gt11. The 2.2-kilobase cDNA insert hybridized to a single 2.6-kilobase poly(A)+ RNA species present in extracts of bovine retina but not of bovine heart, liver, or brain. The nucleotide sequence of the cDNA revealed an open reading frame long enough to encode the entire 39-kDa T alpha polypeptide. The polypeptide sequence deduced from the cDNA would be composed of 350 amino acids and have a molecular weight of 39,971. Portions of the sequence matched reported amino acid sequences of T alpha tryptic fragments, including sites specifically ADP-ribosylated by cholera and pertussis toxins. The predicted sequence also includes four segments, ranging from 11 to 19 residues in length, that exhibit significant homology to sequences of GTP-binding proteins, including the ras proteins of man and yeast and the elongation factors of ribosomal protein synthesis in bacteria, EF-G and EF-Tu. In combination with previous functional studies of tryptic fragments of T alpha, the deduced amino acid sequence makes it possible to predict which portions of the polypeptide interact with other molecules involved in retinal phototransduction.

A general upstream binding factor for genes of the yeast translational apparatus.

Abstract: Fractionation of yeast extracts on heparin-agarose revealed the presence of a DNA footprinting activity that interacted specifically with the 5'-upstream region of TEF1 and TEF2 genes coding for the protein synthesis elongation factor EF-1 alpha, and of the ribosomal protein gene RP51A. The protected regions encompassed the conserved sequences 'HOMOL1' (AACATC TA CG T A G CA) or RPG-box (ACCCATACATT TA) previously detected 200-400 bp upstream of most of the yeast ribosomal protein genes examined. Two types of protein-DNA complexes were separated by a gel electrophoresis retardation assay. Complex 1, formed on TEF1, TEF2 and RP51A 5'-flanking region, was correlated with the protection of a 25-bp sequence. Complex 2, formed on TEF2 or RP51A probes at higher protein concentrations, corresponded to an extended footprint of 35-40 bp. The migration characteristics of the protein-DNA complexes and competition experiments indicated that the same component(s) interacted with the three different promoters. It is suggested that this DNA factor(s) is required for activation and coordinated regulation of the whole family of genes coding for the translational apparatus.

Identification of a nuclear localization signal of a yeast ribosomal protein.

Abstract: To identify a signal involved in transporting a ribosomal protein to the nucleus, we constructed hybrid genes encoding amino-terminal segments of yeast ribosomal protein L3 joined to the amino-terminal end of the entire Escherichia coli beta-galactosidase molecule. The subcellular locations of the corresponding hybrid proteins in yeast were determined by in situ immunofluorescence. The first 21 amino acids of L3 were sufficient to localize beta-galactosidase to the nucleus. This region shows limited homology to portions of other nuclear proteins identified as essential for their transport. Larger fusion proteins were also localized to the nucleus. However, a hybrid protein containing all but the 14 carboxyl-terminal amino acids from L3 initially failed to localize; this defect was corrected by inserting a glycine- and proline-containing bridge between the L3 and beta-galactosidase moieties. The renovated protein was able to associate with ribosomes, suggesting that, in addition to entering the nucleus, this hybrid polypeptide was assembled into 60S ribosomal subunits that were subsequently exported to the cytoplasm.

Identification of ribosomal protein autoantigens.

Abstract: Approximately 20% of patients with systemic lupus erythematosus and with anti-Sm autoantibodies synthesize autoantibodies, called anti-rRNP, to components of the ribosome. We found that anti-rRNP sera reacted predominantly with three ribosomal phosphoproteins of approximate Mr = 38,000, 16,000 and 15,000, both by immunoprecipitation and by immunoblotting. The human autoantibodies cross-reacted with similar antigens present in rodent, brine shrimp, and yeast cells but reacted weakly if at all with proteins of bacteria. Thus the human autoantibodies recognize epitopes that are widely conserved in evolution. Purified ribosomal proteins together with specific rabbit antisera were used to identify the two smaller rRNP antigens as the acidic phosphoproteins of the large ribosomal subunit, designated P1/P2(L40/L41) (rat), eL7/eL12 (Artemia, brine shrimp), and A1/A2 (yeast). These proteins function in the elongation step of protein synthesis in an analogous fashion to the L7/L12 ribosomal proteins of E. coli. The 38,000-dalton rRNP antigen corresponds to a nonacidic protein also associated with the large ribosomal subunit. The human autoantibodies appear to have a specificity similar to that of a previously described mouse monoclonal antibody obtained from mice injected with heterologous (chick) ribosomes, suggesting that both the human polyclonal autoantibodies and the mouse monoclonal recognize a class of epitope(s) that is common in all three ribosomal proteins. In addition, we found that many of the anti-ribosomal sera contained a further class of autoantibodies reactive with naked RNA. These may be similar to the anti-RNA antibodies previously described in both humans and mice with autoimmune disease.

Structure of the Escherichia coli S10 ribosomal protein operon.

Abstract: The complete structure of the Escherichia coli S10 ribosomal protein operon is presented. Based on the DNA sequence, the deduced order of the 11 genes in the operon is rpsJ, rplC, rplD, rplW, rplB, rpsS, rplV, rpsC, rplP, rpmC, rpsQ. The estimated transcribed length of the operon is 5181 base pairs. Putative sequences involved in ribosome binding are discussed. The DNA sequence data corrects several errors in previously determined protein sequence data.

Conserved sequences upstream of yeast ribosomal protein genes.

Abstract: Computer analysis has previously revealed the presence of a 12-nucleotide common sequence element (AACATC CA TG T A G CA; HOMOL1) in the upstream regions of several yeast ribosomal protein genes. By extending the sequence analysis of the 5′-flanking regions of a number of other ribosomal protein genes (including those encoding S10-1, S10-2, S33 and L16-2) we could establish that HOMOL1 occurs upstream of most but not all yeast ribosomal protein genes. Apart from HOMOL1 an additional conserved sequence (ACCCATACATT A T ; RPG-box) was detected in front of nearly all yeast ribosomal protein genes, although in some cases it is present in the opposite orientation in the other strand. There seems to be no correlation between the occurrence of one box and that of the other. However when both boxes are present the RPG-box is always located 3′ to the HOMOL1-sequence mostly at a distance of only a few nucleotides. A further one-to-one comparison of the upstream regions of several yeast ribosomal protein genes revealed extensive additional sequence homologies that are suggested to be involved in the coordinate control of ribosomal protein gene expression in yeast.

Effect of RP51 gene dosage alterations on ribosome synthesis in Saccharomyces cerevisiae.

Abstract: The Saccharomyces cerevisiae ribosomal protein rp51 is encoded by two interchangeable genes, RP51A and RP51B. We altered the RP51 gene dose by creating deletions of the RP51A or RP51B genes or both. Deletions of both genes led to spore inviability, indicating that rp51 is an essential ribosomal protein. From single deletion studies in haploid cells, we concluded that there was no intergenic dosage compensation at the level of mRNA abundance or mRNA utilization (translational efficiency), although phenotypic analysis had previously indicated a small compensation effect on growth rate. Similarly, deletions in diploid strains indicated that no strong mechanisms exist for intragenic dosage compensation; in all cases, a decreased dose of RP51 genes was characterized by a slow growth phenotype. A decreased dose of RP51 genes also led to insufficient amounts of 40S ribosomal subunits, as evidenced by a dramatic accumulation of excess 60S ribosomal subunits. We conclude that inhibition of 40S synthesis had little or no effect on the synthesis of the 60S subunit components. Addition of extra copies of rp51 genes led to extra rp51 protein synthesis. The additional rp51 protein was rapidly degraded. We propose that rp51 and perhaps many ribosomal proteins are normally oversynthesized, but the unassembled excess is degraded, and that the apparent compensation seen in haploids, i.e., the fact that the growth rate of mutant strains is less depressed than the actual reduction in mRNA, is a consequence of this excess which is spared from proteolysis under this circumstance.

Localization of ribosomal protein S1 in the granular component of the interphase nucleolus and its distribution during mitosis.

Abstract: Using antibodies to various nucleolar and ribosomal proteins, we define, by immunolocalization in situ, the distribution of nucleolar proteins in the different morphological nucleolar subcompartments. In the present study we describe the nucleolar localization of a specific ribosomal protein (S1) by immunofluorescence and immunoelectron microscopy using a monoclonal antibody (RS1-105). In immunoblotting experiments, this antibody reacts specifically with the largest and most acidic protein of the small ribosomal subunit (S1) and shows wide interspecies cross-reactivity from amphibia to man. Beside its localization in cytoplasmic ribosomes, this protein is found to be specifically localized in the granular component of the nucleolus and in distinct granular aggregates scattered over the nucleoplasm. This indicates that ribosomal protein S1, in contrast to reports on other ribosomal proteins, is not bound to nascent pre-rRNA transcripts but attaches to preribosomes at later stages of rRNA processing and maturation. This protein is not detected in the residual nucleolar structures of cells inactive in rRNA synthesis such as amphibian and avian erythrocytes. During mitosis, the nucleolar material containing ribosomal protein S1 undergoes a remarkable transition and shows a distribution distinct from that of several other nucleolar proteins. In prophase, the nucleolus disintegrates and protein S1 appears in numerous small granules scattered throughout the prophase nucleus. During metaphase and anaphase, a considerable amount of this protein is found in association with the surfaces of all chromosomes and finely dispersed in the cell plasm. In telophase, protein S1-containing material reaccumulates in granular particles in the nucleoplasm of the newly formed nuclei and, finally, in the re-forming nucleoli. These observations indicate that the nucleolus-derived particles containing ribosomal protein S1 are different from cytoplasmic ribosomes and, in the living cell, are selectively recollected after mitosis into the newly formed nuclei and translocated into a specific nucleolar subcompartment, i.e., the granular component. The nucleolar location of ribosomal protein S1 and its rearrangement during mitosis is discussed in relation to the distribution of other nucleolar proteins.

Emetine resistance of Chinese hamster cells: structures of wild-type and mutant ribosomal protein S14 mRNAs.

Abstract: The Chinese hamster ovary (CHO) cell 40S ribosomal subunit protein S14 provides a unique opportunity to investigate an important mammalian housekeeping gene and its mRNA and protein products. The S14 gene appears to be single copy, and CHO cell S14 mutants have been isolated as emetine-resistant (emtB) clones in tissue culture. Thus, S14 is the only mammalian ribosomal protein whose gene structure and function are amenable to straightforward genetic and biochemical analysis. Recently, we isolated a wild-type Chinese hamster lung cell cDNA clone, pCS14-1, including an almost complete copy of the ribosomal protein S14 message (N. Nakamichi, D. D. Rhoads, and D. J. Roufa, J. Biol. Chem. 258: 13236-13242, 1983). Here we describe comparable cDNAs from wild-type and emtB CHO cells. We report both mRNA and polypeptide sequences of the wild-type and mutant ribosomal protein transcripts. As a consequence of the genetic methods used to obtain our emetine-resistant mutants, the emtB S14 cDNAs differ from wild-type cDNA by single-base changes. Physical and chemical features of polypeptides encoded by the cDNAs are consistent with well-characterized S14 protein polymorphisms. The three emtB mutations analyzed affect two adjacent arginine codons within the very basic S14 carboxyl region, indicating a significant role for this portion of the protein in the function and architecture of the mammalian 40S ribosomal subunit.

Nucleotide sequence of the alpha ribosomal protein operon of Escherichia coli.

Abstract: In Escherichia coli some 19 transcription units encoding the 52 ribosomal proteins are scattered throughout the genome. One of the units, the alpha operon, encodes genes for the ribosomal proteins S13, S11, S4 and L17 as well as the alpha subunit of RNA polymerase. We report here the complete 3.0 kb nucleotide sequence of the alpha operon. In addition, we have determined by S1 nuclease mapping the site of transcription termination in this operon.

Evidence for the exchangeability of acidic ribosomal proteins on cytoplasmic ribosomes in regenerating rat liver.

Abstract: We purified acidic ribosomal proteins (P1 and P2) in good yield from rat liver ribosomes by precipitation of ribosomes with MgCl2 prior to ethanol extraction and chromatography of the extract on a column of CM-cellulose at pH 4.8. The newly-synthesized acidic ribosomal proteins in regenerating rat liver, labeled in vivo with [3H]leucine, were rapidly incorporated into cytoplasmic ribosomes without any detectable time lag and, after reaching a maximum at 30 min, they gradually disappeared from the ribosomes, suggesting a short metabolic-life. However, it was found later that they were re-incorporated slowly when newly-labeled proteins were "chased" by an injection of a large amount of cold leucine intraperitoneally at 15 min after the injection of [3H]leucine. Furthermore, in a long-term experiment, acidic ribosomal proteins were found to disappear with a half-life of 100 h from the ribosomes. Thus, these results suggest that acidic ribosomal proteins have a long metabolic life and are exchangeable on cytoplasmic ribosomes in regenerating rat liver.

Conformation of yeast 18S rRNA. Direct chemical probing of the 5′ domain in ribosomal subunits and in deproteinized RNA by reverse transcriptase mapping of dimethyl sulfate-accessible sites

Abstract: The structure of the 5' domain of yeast 18S rRNA has been probed by dimethyl sulfate (DMS), either in "native" deproteinized molecules or in the 40S ribosomal subunits. DMS-reacted RNA has been used as a template for reverse transcription and a large number of reactive sites, corresponding to all types of bases have been mapped by a primer extension procedure, taking advantage of blocks in cDNA elongation immediately upstream from bases methylated at atom positions involved in the base-pair recognition of the template. Since the same atom positions are protected from DMS in base-paired nucleotides, the secondary structure status of each nucleotide can be directly assessed in this procedure, thus allowing to evaluate the potential contribution of proteins in modulating subunit rRNA conformation. While the DMS probing of deproteinized rRNA confirms a number of helical stems predicted by phylogenetic comparisons, it is remarkable that a few additional base-pairings, while proven by the comparative analysis, appear to require the presence of the bound ribosomal subunit proteins to be stabilized.

Isolation of the SUP45 omnipotent suppressor gene of Saccharomyces cerevisiae and characterization of its gene product.

Abstract: The Saccharomyces cerevisiae SUP45+ gene has been isolated from a genomic clone library by genetic complementation of paromomycin sensitivity, which is a property of a mutant strain carrying the sup45-2 allele. This plasmid complements all phenotypes associated with the sup45-2 mutation, including nonsense suppression, temperature sensitivity, osmotic sensitivity, and paromomycin sensitivity. Genetic mapping with a URA3+-marked derivative of the complementing plasmid that was integrated into the chromosome by homologous recombination demonstrated that the complementing fragment contained the SUP45+ gene and not an unlinked suppressor. The SUP45+ gene is present as a single copy in the haploid genome and is essential for viability. In vitro translation of the hybrid-selected SUP45+ transcript yielded a protein of Mr = 54,000, which is larger than any known ribosomal protein. RNA blot hybridization analysis showed that the steady-state level of the SUP45+ transcript is less than 10% of that for ribosomal protein L3 or rp59 transcripts. When yeast cells are subjected to a mild heat shock, the synthesis rate of the SUP45+ transcript was transiently reduced, approximately in parallel with ribosomal protein transcripts. Our data suggest that the SUP45+ gene does not encode a ribosomal protein. We speculate that it codes for a translation-related function whose precise nature is not yet known.

Selective translational regulation of ribosomal protein gene expression during early development of Drosophila melanogaster.

Abstract: We have previously characterized a cloned cDNA coding for a developmentally regulated mRNA in Drosophila melanogaster whose expression is selectively regulated at the translational level during oogenesis and embryogenesis. In this report we show that this translationally regulated mRNA (rpA1) codes for an acidic ribosomal protein. Furthermore, our results indicate that most ribosomal protein mRNAs are regulated similarly to rpA1 mRNA. This conclusion is based on cell-free translation of mRNAs derived from polysomes and postpolysomal supernatants as well as in vivo labeling experiments. Thus, the translation of many ribosomal protein mRNAs appears to be temporally related to the synthesis of rRNA during D. melanogaster development. The relationship between rRNA transcription and ribosomal protein mRNA translation was further investigated by genetically reducing rRNA synthesis with the use of bobbed mutants. Unexpectedly, neither ribosomal protein mRNA abundance nor translation was altered in these mutants.

The complete primary structure of ribosomal proteins L1, L14, L15, L23, L24 and L29 from Bacillus stearothermophilus.

Abstract: The amino acid sequences of ribosomal proteins L1, L14, L15, L23, L24 and L29 from Bacillus stearothermophilus have been completely determined. This has been achieved by sequence analyses of peptides derived from enzymatic digestions of the proteins with trypsin, chymotrypsin, pepsin, Staphylococcus aureus protease, and Armillaria mellea protease as well as by chemical cleavage with hydroxylamine and cyanogen bromide. Based on the primary structures of the six proteins, their secondary structures were predicted using four different computer prediction programs. A comparison of the amino acid sequences of the studied proteins from B. stearothermophilus with the homologous proteins from Escherichia coli revealed that in four proteins (L1, L15, L24 and L29) between 40–50% of the residue in the sequences are identical, whereas this value is significantly higher (69%) for L14 and lower (28%) for L23. The distribution of those amino acid residues which are identical in the corresponding proteins from the two bacteria is not random along the protein chain: some regions are highly conserved whereas others are not. This finding indicates that the regions which are conserved during evolution are important for the spatial structure and/or function of the protein.

Translational regulation of mRNAs for ribosomal proteins during early Drosophila development.

Abstract: In Drosophila, the vast majority of mRNAs that are polysome associated during oogenesis are also polysome associated during early embryogenesis. We have previously identified an exceptional mRNA that appears to be depleted from early-embryo polysomes [Fruscoloni, P., Al-Atia, G. R., & Jacobs-Lorena, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3359-3363]. This mRNA has been subsequently identified as coding for a ribosomal protein (r-protein) [Kay, M., & Jacobs-Lorena, M. (1985) Mol. Cell. Biol. (in press)]. Changes in association with polysomes of two r-protein mRNAs during early Drosophila development were investigated for this report. Hybridization of cloned DNA probes to blots of RNA obtained from sucrose gradient fractions reveals that r-protein mRNAs are substantially associated with polysomes during oogenesis, depleted from polysomes during early embryogenesis, and again polysome associated during late embryogenesis. Thus, translation of r-protein mRNAs parallels transcription of ribosomal RNA (rRNA) during this time of development. By contrast, no such differences were observed when actin and histone probes were used as controls and hybridized to the same blots. The abundance of mRNAs for r-proteins as a function of development was also measured. Abundance was relatively high and constant during oogenesis and embryogenesis (when translational regulation is apparent), somewhat decreased in larval and pupal stages, and low in adult nonovarian tissues. Coordination between r-protein and rRNA synthesis appears to be achieved by regulating translation of r-protein mRNAs in early embryos and by decreasing their abundance in adult tissues.

Involvement of ribosomal protein L7/L12 in control of translational accuracy

Abstract: The effects of two mutations, which map at the rplL locus and both give a changed 50S ribosomal protein L7/L12, were studied. Both mutations are associated with an increased misreading of all three nonsense codons in vivo and ribosomes from the mutants give an increased misreading of the phenylalanine codon UUU by tRNALeu in vitro. The rplL-associated misreading in vitro is not limited to a particular type of mRNA or tRNA. Results from a translational proofreading assay, using mutant ribosomes, suggest that protein L7/L12 is involved in the control of translational accuracy by contributing to the efficiency of a translational proofreading step(s).

3 16 S Ribosomal RNA Oligonucleotide Cataloguing

Abstract: Publisher Summary This chapter discusses 16 S ribosomal ribonucleic acid (RNA) oligonucleotide cataloguing. 16 S ribosomal RNA is currently the subject of intense study. As a major component of the small ribosomal subunit, elucidation of 16 S rRNA secondary and tertiary structures is a key objective in the ongoing efforts to understand the structure of ribosomes. As a readily isolatable and ubiquitous macromolecule, 16 S rRNA is ideal for studies in molecular systematics. Indeed, in this latter context, studies of 16 S rRNA have become a major factor in the revolution in microbial systematics that is now underway. Progress in both areas is dependent on the availability of sequence data from 16 S rRNAs isolated from a large variety of microbial species. In 16 S rRNA cataloguing the RNA is digested completely with a ribonuclease of known base specificity (usually T 1 , which is specific for guanosine). The resulting oligonucleotide products are separated in two dimensions and visualized by autoradiography. The photographic image of the separation is referred to as the primary fingerprint.

Selective Translational Regulation ofRibosomal Protein Gene Expression during EarlyDevelopment ofDrosophila melanogaster

Abstract: We havepreviously characterized a cloned cDNA coding fora developmentally regulated mRNA in Drosophila melanogaster whoseexpression isselectively regulated atthetranslational level during oogenesis and embryogenesis. Inthis report we showthatthis translationally regulated mRNA (rpAl) codesforan acidic ribosomal protein. Furthermore, our results indicate thatmostribosomal protein mRNAs are regulated similarly torpAlmRNA.Thisconclusion isbased on cell-free translation ofmRNAsderived frompolysomes andpostpolysomal supernatants as well as invivolabeling experiments. Thus,thetranslation ofmany ribosomal protein mRNAsappearstobetemporally related tothesynthesis ofrRNAduring D.melanogaster development. Therelationship between rRNAtranscription andribosomal protein mRNA translation was further investigated bygenetically reducing rRNAsynthesis withtheuseofbobbedmutants. Unexpectedly, neither ribosomal protein mRNA abundance nor translation was altered inthese mutants. Inbothunfertilized seaurchin andXenopus eggs,less than 2%oftheribosomes areassociated withpolysomes andmost mRNAs arefoundinnontranslated messengerribonucleoprotein complexes (mRNPs). After fertilization, thebulkof thesemRNPs are recruited intopolysomes (9,50,53). Recruitment andtranslation ofthesemRNPsisregarded as a common phenomenon rather thana selective processfor specific mRNAs (9). Translation ofmRNA indeveloping Drosophila melanogaster isregulated inadifferent manner. Duringoogenesis andembryogenesis thepercentage of ribosomes inpolysomes remains constant atabout56%, while theproportion ofmRNAs associated withpolysomes remains constant atapproximately 70%(26). Theproportion ofhistone andactin mRNAs associated withpolysomes has beendetermined byRNA blothybridization analysis to remainatabout60% to80% throughout oogenesis and embryogenesis (41,41a). Thebulkofthenonpolysomal mRNA sequences(found as postpolysomal mRNPs)representa subsetofthepolysome-associated mRNAs (11). Moreover, protein synthesis studies haveshownthatthe same abundant andmoderately abundant proteins are synthesized throughout development (24,42,45).Takentogether, theseresults indicate thatthereislittle variation in thepopulation ofabundant mRNA sequencesrepresented on polysomes during D.melanogaster development. Therearehowever, examples ofmRNA sequencesthat arespecifically regulated atthetranslational level during D. melanogaster development. Sucrose gradient fractionation ofpostpolysomal andpolysomal mRNAs hasrevealed that a small numberofmRNA sequencesexist inoocytesthat are present exclusively inthepostpolysomal fraction asmRNPs. Inearly embryos, these sequencesassociate withpolysomes (28), wherethey arepresumed tobeactively translated. We haverecently described (11)thecloning ofa developmentally regulated mRNA sequence(TimRNA)whichistranslationally regulated during Drosophila development ina manner opposite tothatdescribed byMermodetal.(28). Thissequenceisrepresented on polysomes during oogenesis butisselectively excluded frompolysomes during early embryogenesis, ata timewhenrRNA transcription islow.

DNA sequencing of the Escherichia coli ribonuclease III gene and its mutations.

Abstract: A 0.7 kb DNA fragment of the Escherichia coli K12 chromosome was shown to contain the structural gene for RNAse III (rnc). The DNA sequence of the gene was determined and its alteration in an RNAse III defective mutant, AB301-105, was identified. DNA sequence analysis also showed that a secondary-site suppressor of a temperature-sensitive mutation in the E. coli ribosomal protein gene, rpsL, occurred within the rnc gene, providing genetic evidence for the interaction of ribosomal proteins with RNAse III, which in turn acts on the nascent ribosomal RNA during assembly of ribosomes in E. coli.

The genes for yeast ribosomal proteins S24 and L46 are adjacent and divergently transcribed

Abstract: Unlike most yeast ribosomal protein genes studied so far the genes coding for S24 and L46 are adjacent on the genome Sequence analysis showed that the two genes are transcribed divergently, their initiation codons being 630 bp apart Taking the respective ATG translation start sites as reference points, the 5'- end of L46 mRNA was mapped at position -26, while the S24 mRNA showed two major 5'-ends mapping at positions -13 and -16 respectively Unlike most other yeast ribosomal protein genes, the gene for S24 does not contain an intron Its coding region encompasses 390 nucleotides encoding a protein of 14762 D The gene for L46 on the other hand is split by an intron of 386 nucleotides starting after its second codon This gene encodes a small, very basic protein having a molecular weight of 6334 D Yeast ribosomal proteins S24 and L46 show striking homologies with ribosomal proteins from other organisms In particular, yeast L46 is clearly the evolutionary counterpart of rat liver L39 A search of the intergenic region for sequence elements previously identified as common to most yeast ribosomal protein genes, revealed the presence of a single conserved box (RPG-box) roughly equidistant from the transcription initiation sites of both genes We suggest that this box acts as a regulatory signal in either orientation and thus influences the expression of both genes simultaneously

Studies of the GTPase domain of archaebacterial ribosomes

Abstract: Ribosomes from the methanogens Methanococcus vannielii and Methanobacterium formicicum catalyse uncoupled hydrolysis of GTP in the presence of factor EF-2 from rat liver (but not factor EF-G from Escherichia coli). In this assay, and in poly(U)-dependent protein synthesis, they were sensitive to thiostrepton. In contrast, ribosomes from Sulfolobus solfataricus did not respond to factor EF-2 (or factor EF-G) but possessed endogenous GTPase activity, which was also sensitive to thiostrepton. Ribosomes from the methanogens did not support (p)ppGpp production, but did appear to possess the equivalent of protein L11, which in E. coli is normally required for guanosine polyphosphate synthesis. Protein L11 from E. coli bound well to 23S rRNA from all three archaebacteria (as did thiostrepton) and oligonucleotides protected by the protein were sequenced and compared with rRNA sequences from other sources.