Showing papers on "Ribosomal protein published in 1980"

The structure of the yeast ribosomal RNA genes. I. The complete nucleotide sequence of the 18S ribosomal RNA gene from Saccharomyces cerevisiae

Abstract: The cloned 18 S ribosomal RNA gene from Saccharomyces cerevisiae have been sequenced, using the Maxam-Gilbert procedure. From this data the complete sequence of 1789 nucleotides of the 18 S RNA was deduced. Extensive homology with many eucaryotic as well as E. coli ribosomal small subunit rRNA (S-rRNA) has been observed in the 3'-end region of the rRNA molecule. Comparison of the yeast 18 S rRNA sequences with partial sequence data, available for rRNAs of the other eucaryotes provides strong evidence that a substantial portion of the 18 S RNA sequence has been conserved in evolution.

Feedback regulation of ribosomal protein gene expression in Escherichia coli: structural homology of ribosomal RNA and ribosomal protein MRNA

Abstract: Certain ribosomal proteins (r proteins) in Escherichia coli, such as S4 and S7, function as feedback repressors in the regulation of r-protein synthesis. These proteins inhibit the translation of their own mRNA. The repressor r proteins so far identified are also known to bind specifically to rRNA at an initial stage in ribosome assembly. We have found structural homology between the S7 binding region on 16S rRNA and a region of the mRNA where S7 acts as a translational repressor. Similarly, there is structural homology between one of the reported S4 binding regions on 16S rRNA and the mRNA target site for S4. The observed homology supports the concept that regulation by repressor r proteins is based on competition between rRNA and mRNA for these proteins and that the same structural features and of the r proteins are used in their interactions with both rRNA and mRNA.

In vitro expression of Escherichia coli ribosomal protein genes: autogenous inhibition of translation

Abstract: Escherichia coli ribosomal protein L1 (0.5 micro M) was found to inhibit the synthesis of both proteins of the L11 operon, L11 and L1, but not the synthesis of other proteins directed by lambda rifd 18 DNA. Similarly, S4 (1 micro M) selectively inhibited the synthesis of three proteins of the alpha operon, S13, S11, and S4, directed by lambda spcI DNA or a restriction enzyme fragment obtained from this DNA. S8 (3.6 micro M) also showed preferential inhibitory effects on the synthesis of some proteins encoded in the spc operon, L24 and L5 (and probably S14 and S8), directed by lambda spcl DNA or a restriction enzyme fragment carrying the genes for these proteins. The inhibitory effect of L1 was observed only with L1 and not with other proteins examined, including S4 and S8. Similarly, the effect of S4 was not observed with L1 or S8, and that of S8 was not seen with L1 or S4. Inhibition was shown to take place at the level of translation rather than transcription. Thus, at least some ribosomal proteins (L1 S4, and S8) have the ability to cause selective translational inhibition of the synthesis of certain ribosomal proteins whose genes are in the same operon as their own. These results support the hypothesis that certain free ribosomal proteins not assembled into ribosomes act as "autogenous" feedback inhibitors to regulate the synthesis of ribosomal proteins.

Feedback regulation of ribosomal protein gene expression in Escherichia coli.

Abstract: The structural genes for Escherichia coli ribosomal protein (r-protein) genes L1, S4, and S11 were inserted into a plasmid vector containing the lac operator and promoter such that the synthesis of L1, S4, and S11 was controlled by lac regulatory elements. Synthesis of L1, S4, and S11 was stimulated by addition of an inducer of the lac operon (isopropyl thiogalactoside) to exponentially growing cells. Elevated synthesis of L1 caused a specific decrease in L11 synthesis, whereas overproduction of S4 resulted in lowered synthesis of S13 and L17. Stimulation of L1 or S4 synthesis also inhibited cell growth. Overproduction of S11 did not affect synthesis of other r-proteins or alter growth. These results confirm previous in vitro studies [Yates, J. L., Arfsten, A. E. & Nomura, M. (1980) Proc. Natl. Acad. Sci. USA 77, 1837-1841] and support the hypothesis that certain r-proteins have the capacity to selectively inhibit synthesis of r-proteins whose genes are in the same operon as their own.

Crystal structure of a ribosomal component at 2.6 A resolution.

Abstract: Structural information about the ribosomes has been sought in many ways1,2. It is known that the 50S subunit of Escherichia coli ribosomes contains four copies of the ribosomal protein L7/L12 (refs 3–7) in a unique region8,9. A complex can be isolated consisting of those four copies bound to another ribosomal protein, L10 (refs 10–13). L7/L12 aggregates into elongated dimers in solution14,15. Ribosomes deprived of L7/L12 have a reduced affinity for supernatant factors such as EF-Tu and EF-G and almost no capacity for factor-dependent GTP hydrolysis16–18. The sequence of the 120 amino acid residues in L7/L12 from E. coli has been determined19, and two domains of L7/L12 (residues 1–36 and 53–120, respectively) have been crystallized separately20. We now report the first crystal structure of a ribosomal component, a C-terminal fragment (CTF) of the protein L7/L12 from E. coli, determined to 2.6 A resolution. The CTF has a compact, plum-shaped tertiary structure with a high content of secondary structure. The three α-helices and three β-strands in CTF account for 32% α-helix and 14% β-structure in the intact L7/L12 protein. A binding site for anions shows some resemblance to the known nucleotide binding site in many proteins.

Isolation and mapping of a cloned ribosomal protein gene of Drosophila melanogaster

Abstract: Molecular cloning techniques are particularly well suited to the study of gene organization in Drasophila melanogaster because recombinant DNA can easily be localized in the genome by in situ hybridization to salivary gland polytene chromosomes. We report here the isolation and preliminary characterization of a recombinant phage, designated C25, containing a bona fide D. melanogaster ribosomal protein gene. In situ hybridization demonstrates that this sequence maps to region 99D on chromosome 3.

Insulin-treated 3T3-L1 adipocytes and cell-free extracts derived from them incorporate 32P into ribosomal protein S6.

Abstract: Differentiated 3T3-L1 preadipocytes preincubated for 60 min with 32Pi incorporate 32P into ribosomal protein S6 within 5 min after exposure to 0.1-1.0 nM insulin. Undifferentiated 3T3-L1 cells, which possess only 3-5% of the high-affinity cell surface insulin receptors present on the differentiated cells, are less sensitive to this stimulation. Under the same conditions used for insulin, epidermal growth factor, antibody to the insulin receptor, and a combination of isoproterenol and 1-methyl-3-isobutylxanthine also promote 32P incorporation into S6 in the differentiated cells although less effectively than insulin. Cell-free extracts derived from cells treated for 5-10 min with either physiological concentrations of insulin or epidermal growth factor (0.1 microgram/ml) reflect intact cells and catalyze the incorporation of 32P from exogenous [gamma-32P]ATP into ribosomal protein S6.

Functional Studies on Ribosomes Lacking Protein L1 from Mutant Escherichia coli

Abstract: The function of protein L1 in protein biosynthesis has been examined using ribosomes from two independently derived mutants of Escherichia coli, both of which lacked this protein In systems in vitro with phage MS2 RNA or poly(U) as message, the mutant ribosomes showed from 40% to 60% of the activity of wild-type ribosomes The reduction in activity was apparent in the kinetics of [14C]phenylalanine incorporation throughout the incubation period The activities were restored fully to the wild-type level by the addition of purified protein L1 These results show on the one hand that protein L1 is not essential for protein biosynthesis but, on the other, its presence can significantly increase the overall rate of this process The data further indicate it as likely that protein L1 exerts its effect at the elongation step

Autogenous control of Escherichia coli ribosomal protein L10 synthesis in vitro

Abstract: The DNA-dependent in vitro synthesis of Escherichia coli ribosomal protein L10 was inhibited when L10 was added to the protein-synthesizing incubations. Addition of L10 had little or no effect on the synthesis of ribosomal protein L12, elongation factor Tu (tufB), or the beta and beta' subunits of RNA polymerase. In addition, ribosomal protein L12 did not inhibit its own synthesis or the synthesis of L10. Experiments using a mRNA-directed system showed that the inhibition of the synthesis of L10 by itself is at the level of translation of protein synthesis. The mechanism of inhibition does not appear to be due to increased degradation of L10 mRNA.

Sequence homologies among E. coli ribosomal proteins: evidence for evolutionarily related groupings and internal duplications.

Abstract: The complete or partial sequences of 47E. coli ribosomal proteins described in the literature have been examined by computerized search and matching programs. In contrast to results previously reported by other investigators, sequence homologies were uncovered among some of these ribosomal proteins that are well beyond statistical expectations. Moreover, alignments of the most strongly homologous sequences suggested the existence of a network of family groupings. Several of these proteins also exhibit internal homologies, indicating that they have been elongated by a series of tandem duplications.

Interaction Between Aminoglycoside Uptake and Ribosomal Resistance Mutations

Abstract: Mutants resistant to the 2-deoxystreptamine aminoglycosides hygromycin B and gentamicin were analyzed biochemically and genetically. In hygromycin B-resistant strains, ribosomal alterations were not detectable by electrophoretic or genetic experiments. Rather, as was demonstrated for one strain in detail, resistance to this drug seems to be the consequence of several mutations, each impairing drug accumulation, namely of a deletion of a gene close to the proC marker which potentiates the effect of a second mutation in the unc gene cluster. Three mutants resistant to gentamicin which were previously demonstrated to harbor an altered ribosomal protein, L6, were shown in addition to contain unc . Both the unc and the ribosomal mutation greatly impair the drug accumulation ability of the mutants. Further evidence for the direct effect of ribosomal mutations on the uptake of aminoglycosides was obtained with strains that possess ribosomes with increased affinity for dihydrostreptomycin. Dihydrostreptomycin transport by these cells is greatly stimulated; thus, the hypersensitivity of these mutants is caused by increased binding affinity for dihydrostreptomycin and its secondary effect on the uptake process. Experiments were also performed on the biochemical basis of the third phase of aminoglycoside transport (acceleration phase). The condition for its onset is that ribosomes are active in protein synthesis irrespective of whether the proteins synthesized are functional. This, and the failure to observe the synthesis of new proteins upon the addition of aminoglycosides, do not support the view of autoinduction of a cognate or related transport system. Images

Protein identifications on O'Farrell two-dimensional gels: locations of 55 additional Escherichia coli proteins.

Abstract: The resolution of proteins from whole-cell homogenates by two-dimensional gel electrophoresis is sufficiently reproducible and precise to permit different laboratories to exchange information about them. To the previous total of 81 we add the locations of 55 Escherichia coli proteins determined with the aid of purified proteins and mutant strains supplied by many investigators. The criteria used to establish the identifications of protein spots include migration with marker proteins, altered position or amount in appropriate mutant or plasmid-carrying strains, physiological behavior, and peptide map pattern.

Ribosomal protein modification in Escherichia coli. II. Studies of a mutant lacking the N-terminal acetylation of protein S18.

Abstract: A mutant of Escherichia coli K12 has been isolated which shows an alteration in the ribosomal protein S18. Genetic analyses have revealed that the mutation causing this alteration maps at 99.3 min of the E. coli genetic map, between dnaC and deo. This indicated that the mutation has occurred in a gene different from the structural gene for this protein which has been located at 94 min. From the N-terminal amino acid sequence analysis it is concluded that the mutation has resulted in loss of the N-terminal acetyl group of this protein. The gene which is affected in this mutant is termed rimI that most likely specifies an enzyme acetylating the N-terminal alanine of protein S18. The mutation does not affect the acetylation of two other ribosomal proteins, S5 and L12, both of which are known to be acetylated in wild-type E. coli K12.

Altered ribosomal protein L29 in a cycloheximide-resistant strain of Saccharomyces cerevisiae.

Abstract: A spontaneous high-level cycloheximide-resistant mutant of the yeast Saccharomyces cerevisiae (strain cy32) is found to have an altered protein of the large subunit (60S) of cytoplasmic ribosomes, namely protein L29. The resistance character segregates together with this biochemical defect and is semidominant in heterozygous diploids. Judged from in vitro susceptibility to inhibition by cycloheximide there are at least 50% resistant ribosomes present in such diploid strains. From these results it is concluded that cycloheximide resistance of mutant cy32 is caused by mutation of a single gene and that it is the structural gene for L29 which is affected.

Purification and Primary Structure Determination of the N‐Terminal Blocked Protein, L11, from Escherichia coli Ribosomes

Abstract: Protein L11 was isolated from the 50-S subunit of Escherichia coli ribosomes, using two salt extractions and two chromatographic separations on CM-cellulose. The unusual behavior of the protein when run on sodium dodecyl sulfate electrophoresis showed multiple bands. The complete primary structure of protein L11 is presented in detail. Its sequence was derived from peptides obtained by digesting the protein with trypsin, chymotrypsin, thermolysin, Staphylococcus aureus protease and, after modification, with trypsin. Chemical cleavage was performed with cyanogen bromide. Sequencing of the various peptides was achieved by manual micro-dansyl-Edman degradations and automatic methods. The N-terminal residue of the protein is blocked and was not degradable in the liquid-phase sequenator by the Edman method. It was identified by a combination of enzymatic cleavage and mass spectrometry. Protein L11 contains three methylated amino acid residues, a Nα-trimethylalanine, and two residues of Nα-trimethyllysine. Their behaviour and influence in the sequence elucidation are described. The protein contains 141 amino acid residues and has a molecular weight of 14874. Secondary structure predictions of the protein are given, and its sequence is compared with those of other E. coli ribosomal proteins.

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.

Abstract: 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.

Autogenous regulation of the synthesis of ribosomal proteins, L10 and L7/12, in Escherichia coli.

Abstract: The in vitro synthesis of Escherichia coli ribosomal proteins, L10 and L7/12, is specifically repressed by the addition of the L10-L7/12 complex, while that of other ribosomal proteins encoded by the neighboring operons is not affected. Thus the expression of the rpoBC operon is controlled by two autorepression systems, one for the two ribosomal proteins and the other for RNA polymerase β and β′ subunits, both operating probably at the translational level.