Showing papers on "Ribosomal protein published in 1975"
TL;DR: Analysis of the double-stranded, S 1 -resistant fragments indicates that there is a direct relationship between the hairpin loops seen in the electron microscope and secondary structure in aqueous solution.
1,231 citations
TL;DR: Here the authors begin to define 16S rRNA function by localising the major conserved regions in the molecule through a comparative analysis of 27 prokaryotic 16 S rRNA primary structures.
Abstract: THERE is no doubt that the large ribosomal RNAs play specific roles in ribosome function1. Yet the thrust of experimentation during the past 5 years indicates clearly that the biologist tends to view these roles as structural, function being reserved by and large for the ribosomal protein components2,3. Whether or not this prejudice can be maintained remains to be seen. In any case, the existence of an approximate sequence for a 16S rRNA provides a basis for detailed characterisations of this molecule3,4. Here we begin to define 16S rRNA function by localising the major conserved regions in the molecule through a comparative analysis of 27 prokaryotic 16S rRNA primary structures.
263 citations
TL;DR: Two copies of the structural gene for the elongation factor EF-Tu have been identified in Escherichia coli: one near rif and the other near str.
Abstract: Two copies of the structural gene for the elongation factor EF-Tu have been identified in Escherichia coli: one near rif and the other near str. The latter seems to belong to a single transcriptional unit together with the genes for ribosomal protein S7, S12 (str) and the elongation factor EF-G (fus).
241 citations
TL;DR: None of the proteins except L7/L12 is present at a level significantly different from one molecule per ribosome, suggesting that the ribosomes of Escherichia coli are homogeneous in vivo.
Abstract: A ribosome preparation from E. coli made without stringent washing procedures has been shown to contain the same relative amounts of nearly all the ribosomal proteins as ribosomes in intact cells. Stoichiometric measurements on all the proteins of this preparation except for L8, L20, L31 and L34 have been made using an isotope dilution technique. When the scatter of the values obtained, the uncertainty in the molecular weights, and the losses occurring during extraction are taken into account, none of the proteins except L7/L12 is present at a level significantly different from one molecule per ribosome. There are multiple copies of L7/L12. These data suggest that the ribosomes of Escherichia coli are homogeneous in vivo.
174 citations
TL;DR: The total number of copies of protein L12 and its α-N-acetylated form L7 in Escherichia coli ribosome has been determined and is found to be four and the observed variation in L 12 L 7 ratio would reflect continuous changes in the relative abundance of these subspecies in vivo during the growth cycle.
170 citations
TL;DR: Eight to ten of the 19 proteins of the 30S subunit have shown antibody attachment sites at remote points on the surface of the ribosome, at distances which are incompatible with globular shapes; these proteins must therefore have elongated or fibrous structures within the Ribosome.
Abstract: Binding sites for antibodies specific to nineteen of the twenty-one ribosomal proteins from the 30S subunit of E. coli ribosomes have been localized on the surface of the 30S ribosomal subunit by immune electron microscopy. The locations of 13 ribosomal proteins from the 50S subunit were similarily assessed. The arrangement of these proteins is illustrated in three-dimensional models of the 30S and 50S ribosomal subunits and of 70S ribosomes. With specific antibodies to six proteins of the 30S subunit we found only one attachment point for each protein. Antibodies against each of nine of the proteins attached at two separate sites that were separated by various distances. Four further proteins were exposed at three or four sites for antibody binding. Altogether eight to ten of the 19 proteins of the 30S subunit have shown antibody attachment sites at remote points on the surface of the ribosome, at distances which are incompatible with globular shapes; these proteins must therefore have elongated or fibrous structures within the ribosome. On the other hand, only two proteins of the 50S subunit, namely L11 and L18, have so far revealed two separated antibody binding sites; proteins L7/L12 occurred, however, at multiple sites.
139 citations
TL;DR: It is suggested that this extraribosomal factor modulates the intrinsic activity of ribosomes to catalyze peptide-bond synthesis, and is regarded as a new factor required for peptide chain elongation, which it is called EF-P.
Abstract: A soluble protein factor was isolated, free of elongation factor (EF)-T and EF-G, based on its ability to stimulate the synthesis of peptide bonds using ribosomal bound 70S-AUG-N-formyl-[35S]methionyl-tRNA complex and added puromycin as substrates. Over 90% of this activity was found in the ribosome-free cytoplasm of Escherichia coli extracts. Otherfeatures such as molecular weight, purification properties, and catalytic activities distinguish this factor from ribosomal proteins and known activators of translation. The factor requires all components needed for peptide bond synthesis and is inhibited by antibiotics known to specifically block the peptidyl transferase activity of ribosomes. The factor increases the binding affinity of the ribosome for the aminoacyl-tRNA analog puromycin about 10-fold. We suggest that this extraribosomal factor modulates the intrinsic activity of ribosomes to catalyze peptide-bond synthesis, and regard it as a new factor required for peptide chain elongation, which we call EF-P.
115 citations
TL;DR: Cytoplasmic nonpolysomal mRNAs have been isolated in the form of 16-40S ribonucleoprotein particles from the postribosomal supernatant of 14-day-old chick embryonic muscles, suggesting that they may have a role in the regulation of translation in developing muscles.
Abstract: Cytoplasmic nonpolysomal mRNAs have been isolated in the form of 16-40S ribonucleoprotein particles from the postribosomal supernatant of 14-day-old chick embryonic muscles. An 8-20S RNA fraction isolated from these particles directs the synthesis of actin in a wheat germ embryo S-30 system, as judged by copurification of the products with chicken muscle actin by repeated cycles of G- to F-actin transformation; mobilities of the purified product on sodium dodecyl sulfate-polyacrylamide gels and urea gels; and analysis of the CNBr-cleaved peptides. The 16-40S particles have a buoyant density of 1.4 g/cm3 which corresponds to an RNA/protein ratio of 1:3. They do not contain detectable levels of ribosomal subunits, as judged by the absence of typical ribosomal proteins in the range of 15,000-30,000. They contain at least eight distinct polypeptide species in the molecular weight range of 44,000-100,000, including a prominent 44,000 species. The presence of these particles suggests that they may have a role in the regulation of translation in developing muscles.
103 citations
TL;DR: The locations of exposed regions of S11, S13 and S19, in combination with the crosslinking results of others, suggest a binding site for initiation factor IF3 on a region of the 30 S surface, comprising the platform, the cleft, part of the partition, and parts of the upper “one third” of the subunit.
102 citations
TL;DR: The finding that the Qbeta host factor is associated with ribosomes in vivo completes the demonstration that all of the host-supplied proteins required for phage Qbeta RNA replication in vitro are either associated with Ribosomes or are involved in the protein-synthetic machinery of the cell.
101 citations
TL;DR: Evidence is presented which indicates that additional proteins interact with the RNA at later stages of subunit assembly, and appears to depend upon both the secondary structure and conformation of the RNA molecule.
Abstract: Specific binding sites for five proteins of the Escherichia coli 30S ribosomal subunit have been located within the 16S RNA. The sites are structurally diverse and range in size from 40 to 500 nucleotides; their functional integrity appears to depend upon both the secondary structure and conformation of the RNA molecule. Evidence is presented which indicates that additional proteins interact with the RNA at later stages of subunit assembly.
TL;DR: The reexamination of the topographic relationship between 30s protein Sl and the 3’-terminus of the 16s RNA was prompted by recent experiments which suggest a well defined functional relationship between these ribosomal components.
TL;DR: Comparison of the rate of synthesis of the r-proteins coded for by genes on the phage and the level of r-protein messenger RNA homologous to these genes during steady-state and transition conditions indicates that the expression ofr-protein genes is controlled primarily by a mechanism which regulates the amount of mRNA for r- protein which is available for translation.
TL;DR: Results can explain why S1 is an essential component of the ribosome for translation of natural mRNA and why aurin tricarboxylic acid blocks initiation.
Abstract: Ribosomal protein S1 reversibly binds the 49-nucleotide fragment that is cleaved from the 3' end of 16S rRNA in ribosomes by colicin E3. The fragment has secondary structure in the form of a hairpin loop. At the base of the stem is a sequence (A-C-C-U-C-C) thought to be involved in the base pairing with complementary sequences in mRNA during the initiation of protein synthesis. The role of S1 may be to stabilize this region of the fragment in an open conformation to allow for base pairing to mRNA. This model is supported by the observation that S1 binds specifically to this region of the fragment. In addition, aurin tricarboxylic acid, an inhibitor of protein synthesis, reverses this effect by disrupting the S1-RNA complex. These results can explain why S1 is an essential component of the ribosome for translation of natural mRNA and why aurin tricarboxylic acid blocks initiation.
TL;DR: Initiation factors for protein synthesis were identified and partially purified from free 40 S subunits, and specific factor activities were demonstrated by initiation complex formation with ribosome subunits and Met-tRNA f in the presence and absence of mRNA.
TL;DR: Of the 18 ribosomal proteins found in the LiC1 SP, only L16 is essential for reconstitution of peptidyl transferase activity, which can be restored to theLiC1 cores by reconstitutes with LiC 1 SP under conditions of high temperature and high ionic strength.
Abstract: Extraction with 2 M lithium chloride removes a group of proteins (LiC1 SP) from 50S ribosomal subunits. Both the LiC1 SP and the resulting cores, which contain the remaining proteins as well as 5S and 23S RNA, lack peptidyl transferase activity, as measured by the "fragment reaction". Activity can be restored to the LiC1 cores by reconstitution with LiC1 SP under conditions of high temperature and high ionic strength. The LiC1 SP proteins were fractionated by carboxymethyl-cellulose and Sephadex G-100, and the individual fractions were tested by this reconstitution system. Of the 18 ribosomal proteins found in the LiC1 SP, only L16 is essential for reconstitution of peptidyl transferase activity.
TL;DR: The transducing phage lambdarifd18 isolated by Kirschbaum and Konrad was found to carry structural genes for several 50S ribosomal proteins and 16S and 23S rRNA and contains a cluster of genes essential for transcription and translation.
Abstract: The transducing phage lambdarifd18 isolated by Kirschbaum and Konrad [(1973 J Bacteriol 116, 517-526] was found to carry structural genes for several 50S ribosomal proteins and 16S and 23S rRNA It has previously been demonstrated [Kirschbaum & Scaife (1974) Mol Gen Genet 132, 193-201] that this phage carries genes for the DNA-dependent RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2776) subunits beta and beta' Thus, the region of the E coli chromosome carried by lambdarifd18 contains a cluster of genes essential for transcription and translation
TL;DR: Mutations in ribosomal protein S5 can act as ram mutations like mutations in protein S4, and are able to derestrict the restriction of translational ambiguity imposed by strA mutations, though to different degrees depending on the type of mutation.
Abstract: The effect of three different types of mutations in ribosomal protein S5 of Escherichia coli on translational fidelity has been studied. Two of them, namely that conferring resistance to spectinomycin and that selected for partial suppression of a temperaturesensitive alanyl-tRNA synthetase mutation, do not exhibit ribosomal ambiguity in the in vivo and in vitro test system employed. In constrast, mutations in ribosomal protein S5 selected for suppression of streptomycin dependence mutations are able to derestrict the restriction of translational ambiguity imposed by strA mutations, though to different degrees depending on the type of mutation. Mutants in which streptomycin dependence is suppressed by an alteration in protein S5 are more restrictive than mutants resistant to streptomycin. Again, the extent of restriction depends on the type of the strA
d allele. In conclusion: mutations in ribosomal protein S5 can act as ram mutations like mutations in protein S4. The part of the S5 polypeptide involved in control of translational fidelity is different from regions altered in spectinomycin resistant strains and in the alanyl-tRNA synthetase suppressor mutant.
TL;DR: The use of the nucleotide sequence data in studies of the ribosomal protein binding sites and data on sequence heterogeneities, repetitions and the location of modified nucleotides are presented.
Abstract: Recent progress in the nucleotide sequence analysis of the 16S ribosomal RNA from E. coli is described. The sequence which has been partially or completely determined so far encompasses 1520 nucleotides, i.e. about 95% of the molecule. Possible features of the secondary structure are suggested on the basis of the nucleotide sequence and data on sequence heterogeneities, repetitions and the location of modified nucleotides are presented. In the accompanying paper, the use of the nucleotide sequence data in studies of the ribosomal protein binding sites is described.
TL;DR: Radioimmunodiffusion with antisera prepared against 20 individual 30 S ribosomal proteins S1, S2, S11, S12, S13, S14, and S19 was interpreted to mean that initiation factor IF-2 was present in covalent cross-linked complexes containing those proteins.
TL;DR: The results indicate lambdadspcl probably carries at least 22 ribosomal protein genes and lamb dadspc2 at least 26 genes, and all these genes are clustered between trkA and strA.
Abstract: Specialized lambda transducing phages have been isolated carrying approximately half the ribosomal protein genes of E. coli. These phages carry regions of the bacterial chromosome between aroE and fus. The ribosomal protein genes on these phages have been identified by the stimulation of ribosomal protein synthesis in ultraviolet-irradiated bacteria following infection by the transducing phage, and by the in vitro synthesis of ribosomal proteins in a DNA-dependent protein synthesizing system. The results indicate lambdadspcl probably carries at least 22 ribosomal protein genes and lambdadspc2 at least 26 genes. All these genes are clustered between trkA and strA. At least 13 of them have not been previously mapped.
TL;DR: The results are consistent with the hypothesis that reticulocyte ribosomes contain one copy of most of their protein constituents, and in agreement with previous estimations, derived from physico-chemical measurements of the total protein in mammalian ribosomal subunits.
TL;DR: Results indicate that rpyL is the structural gene for L7/L12 and that this region of the E. coli chromosome contains a cluster of structural genes for ribosomal proteins.
Abstract: An Escherichia coli mutant, ts9, previously reported by Flaks et al. (Cold Spring Harbor Symp. Quant. Biol. 31, 623-631, 1966) to have an electrophoretically altered ribosomal protein, has been further characterized and the altered component has been identified as L7/L12. Although mutant ts9 is temperature sensitive for growth (rts-), the rts and L7/L12 mutations are genetically separable and are both located between argH and rif. The L7/L12 mutation (rpyL) maps very close to relC, mutants of which have a defect in the 50S ribosomal subunit. The gene order is argH-rts-(rpyL,relC)-rif. Protein synthesis directed by bacteriophage lambdacI857S7drifd18 in ultraviolet-irradiated cells indicates that L7/L12, As well as L1, L10, L11, and possibly L8 or L9 are coded by the phage DNA. Our results indicate that rpyL is the structural gene for L7/L12 and that this region of the E. coli chromosome contains a cluster of structural genes for ribosomal proteins.
TL;DR: Under appropriate conditions, the entire population of E. coli 30S subunits can be isolated as the S1-containing subspecies, and protein S1 is lost by salt treatment of ribosomes.
Abstract: Among several subspecies of 30S subunits of Escherichia coli observed by polyacrylamide-agarose gel electrophoresis, only the slow-moving, protein S1-containing subspecies participates in the formation of the 30S initiation complex with coliphage MS2 RNA as mRNA; the other subspecies retain activity with AUG as mRNA; they are also active in the poly(U)-directed binding of Phe-tRNA. Protein S1 from Caulobacter crescentus substitutes for E. coli S1 despite the fact that C. crescentus ribosomes do not bind MS2 RNA. Under appropriate conditions, the entire population of E. coli 30S subunits can be isolated as the S1-containing subspecies. Protein S1 is lost by salt treatment of ribosomes.
TL;DR: Homopolymer RNA-cellulose chromatography appears to be a simple, general technique, useful for the efficient isolation of a variety of RNA-binding proteins.
TL;DR: Two ribosomes from encysted gastrulae of the brine shrimp Artemia salina contain two acidic proteins, which are homologous to the Escherichia coli proteins L7 and L12, and their functional requirement in the elongation factor dependent binding of aminoacyl transfer RNA to the ribosome is determined.
Abstract: 60S ribosomes from encysted gastrulae of the brine shrimp Artemia salina contain two acidic proteins, which are homologous to the Escherichia coli proteins L7 and L12. The proteins were purified and characterized with respect to molecular weight, amino-acid composition, peptide maps, and their functional requirement in the elongation factor dependent binding of aminoacyl transfer RNA to the ribosome.
TL;DR: Methylated ribosomal proteins from Escherichia coli 50S subunit are localized by growing cells in a medium containing (1-14C)methionine and (3H-methyl)-meth ionine and comparing the 3H/14C ratio for each of the 50S ribosome proteins.
Abstract: Methylated ribosomal proteins from Escherichia coli 50S subunit are localized by growing cells in a medium containing (1-14C)methionine and (3H-methyl)-methionine and comparing the 3H/14C ratio for each of the 50S ribosomal proteins. The following proteins are methylated: L11, L1, L3, L5, L7, L8, L9, L12, L18, and L33. The nature and stoichiometry of the methylated amino acid(s) in each of the methylated proteins are determined. Protein L11 is the most heavily methylated of all the 50S subunit proteins. This protein has previously been implicated in the peptidyl transferase reaction during protein synthesis (K. H. Nierhaus and V. Montejo (1973), Proc. Nat. Acad. Sci. U. S. 47, 1588-1602). Three proteins (L1, L3, and L5) have intermediate levels of methylation and contain about 0.4-0.6 methyl groups each per molecule of protein. Five other proteins (L7, L8, L9, L12, and L18) are also methylated to a slight extent (-0.1 methyl group/molecule of protein). One unknown methylated neutral amino acid was detected in protein L11 and at least one and possibly two other unidentified methylated amino acids appeared to be present in protein L33.
TL;DR: Fourteen proteins from the large subunit of Escherichia coli ribosomes were analyzed in an improved sequenator using a new vacuum system with a recorder, cool traps, and new valves which work free of a dead volume.
Abstract: Fourteen proteins from the large subunit of Escherichia coli ribosomes were analyzed in an improved sequenator. In addition to our previously described modifications of a Beckman sequenator, new valves which work free of a dead volume were constructed. By this and the previous improvements (e.g., a new vacuum system with a recorder, cool traps, automatic conversion) much better results were obtained than before. It was even possible to use (in addition to the standard methods, e.g., thin-layer chromatography and amino acid analysis) mass spectrometry without preceding gas chromatography for identification of the released PTH amino acids. Our experience with the various methods, especially mass spectrometry, is described and the techniques are compared. The results obtained by the described methods on the amino acid sequences of the 14 ribosomal proteins are summarized.
TL;DR: The results indicate that the N-terminal part of L7/L12 is responsible for its ability to bind to 50S ribosomes and that L7 /L12 together with L10 form a protein cluster on the ribosome.
TL;DR: Evidence is presented that in the Ehrlich cell one of the native subunit associated proteins is the mammalian initiation factor that forms a Met-tRNA-f-factor-GTP complex, and is required for the binding of Met- t RNA-f to the 40S subunit.
Abstract: Our previous work has shown that the native 40S ribosomal subunits (those found free in the cell sap) but not polyribosomal 40S subunits have additional associated proteins that are removed by 0.5 M KCl. In this communication we present evidence that in the Ehrlich cell one of the native subunit associated proteins is the mammalian initiation factor that forms a Met-tRNA-f-factor-GTP complex, and is required for the binding of Met-tRNA-f to the 40S subunit. Initial examination of the KCl wash of the Ehrlich cell total ribosomal pellet revealed a factor which (1) shifted the elution of Met-tRNA-f and of GTP from the included to the excluded volume on Sephadex G-100 chromatography, (2) stimulated the binding of Met-tRNA-f to Millipore filters, and (3) stimulated the binding of Met-tRNA-f to salt-washed 40S subunits. These activities were dependent upon or enhanced by GTP; were inhibited by GDP; were much greater for Met-tRNA-f than for Met-tRNA-m or for lysyl-tRNA; and were concentrated in the KCl ribosomal wash and were not detected in the cell soluble fraction. Met-tRNA-f bound in conjunction with a specific amount of KCl wash protein, to form a distinctive particle of bouyant density 1.40 g cm minus 3 in CsCl, identical in density to one form of the native 40S subunit. Native 40S subunits, but no other subunits, contained a factor which was eluted by 0.5 M KCl and which (1) stimulated the binding of Met-tRNA-f to Millipore filters, and (2) stimulated the binding of Met-tRNA-f to salt-washed 40S subunits. The factor appeared to be localized on the native 40S subunit of density 1.40 g cm minus 3.