Showing papers on "Ribosomal protein published in 1974"
TL;DR: It is concluded that the binding of S17 to 16 S RNA is specific and determined the positions of S15, S16, S17, and S12 in the assembly map and have clarified subsequent interactions depending on these proteins.
428 citations
TL;DR: It was shown by two-dimensional polyacrylamide gel electrophoresis that all of the radioactive phosphate incorporated into ribosomes was in a single small subunit protein, S6, which contained increasing numbers of radioactive phosphoserine residues.
280 citations
TL;DR: A two-dimensional electrophoresis system for analysis of ribosomal proteins with several advantages over previous systems is described, and is relatively simple and inexpensive to construct and use.
253 citations
TL;DR: The results indicate that the ratios RNA/DNA, RNA/protein, and protein/DNA give linear relationships with the growth rate, whereas RNA/cell andprotein/cell show a more complex growth rate dependency.
Abstract: By using Escherichia coli B/r, the cellular amounts of ribonucleic acid (RNA) and protein were determined as a function of the steady-state growth rate (0.67 to 2.40 doublings per h) by a method which combines measurements of the RNA to deoxyribonucleic acid (DNA) ratio and the differential rate of ribosomal protein synthesis with the Cooper and Helmstetter theory of DNA replication. The results indicate that the ratios RNA/DNA, RNA/protein, and protein/DNA give linear relationships with the growth rate (above 1.2 doublings per h), whereas RNA/cell and protein/cell show a more complex growth rate dependency. The significance of these relationships is discussed. Finally, a detailed description of the growth parameters and composition of E. coli B/r is presented.
187 citations
TL;DR: Subunit I of the RNA phage-specific Qβ replicase is shown to be identical with the Escherichia coli 30 S ribosomal protein Sl by the following criteria: ability to restore Qβ RNA-directed activity of Qβ Replicase lacking subunit I, immunological cross-reactivity, and identity of the first four amino acids at the NH2 terminus.
159 citations
TL;DR: The structure of ribosomal 5S RNA was determined by Sanger and his associates some time ago; the sequence of nucleotides in 16S RNA has been all but completed and that of 23S RNA is well under way and there is a great likelihood that the primary structure of all the components of prokaryotic ribosomes will soon be known.
Abstract: INTRODUCTION The unraveling of the structure and insight into the details of the function of prokaryotic ribosomes is derived from three technological advances, each an extraordinary achievement: the isolation, purification and characterization of the 55 proteins of E. coli ribosomes (see Wittmann, this volume); the reconstitution of active ribosomal subunits from their molecular components, i.e., from RNA and protein (see Nomura and Held, this volume); and finally the application of immunochemical techniques to their analysis (see Stoffler, this volume). The critical achievement was the separation of the ribosomal proteins; it made everything else (including re-constitution and the immunochemistry) possible. The structure of ribosomal 5S RNA was determined by Sanger and his associates (Brownlee, Sanger and Barrell 1968) some time ago; the sequence of nucleotides in 16S RNA has been all but completed (Fellner et al. 1972) and that of 23S RNA is well under way (see Fellner, this volume). Thus there is a great likelihood that the primary structure of all the components of prokaryotic ribosomes will soon be known. The situation with respect to eukaryotic ribosomes is not nearly so far advanced. The individual proteins of eukaryotic ribosomes have not been isolated (in significant amounts), nor have they been purified or characterized, and that is a major impediment to further progress in analysis of the structure and function of the particles. There is no practical means of reconstituting functional eukaryotic ribosomes from their components, and only very limited use has been made of antibodies to study animal ribosomes. All...
146 citations
TL;DR: The results allowed the proposal of a three-dimensional model of the 50S subunit with the location of six different ribosomal proteins as illustrated in Fig. 13.
Abstract: The location of the ribosomal proteins L14, L17, L18, L19, L22 and L23 on the surface of the 50S subunit of E. coli ribosomes was determined by immune electron microscopy. Antibodies (bivalent IgG's) specific for the six ribosomal proteins were used to form 50S subunit dimers (50S-IgG-50S). The dimers were separated from non-bound antibodies and 50S subunit-monomers and larger aggregates by sucrose density gradient centrifugation. The attachment of each of the six immunoglobulins to 50S subunits was visualized directly by electron microscopy using negative staining and correlated with one or more of the structural features of the particle. Each of the proteins was found to occupy a unique position. Proteins L14, L19 and L23 are located in the region of the 50S subunit which interacts with the 30S particle. Proteins L17 and L22 are on the opposite side of the 50S ribosomal subunit, whilst L18 holds a position on one of the lateral protuberances of a crown-like shaped 50S subunit. Two main forms of 50S subunits were seen on the electron micrographs: one of them had a crown-like shape; the second revealed kidney shaped images. It was demonstrated that these two images are only projections of one structure of the subunit that resembled an armchair; each of the forms could be transformed into the other through rotation by 90°. The results allowed the proposal of a three-dimensional model of the 50S subunit with the location of six different ribosomal proteins as illustrated in Fig. 13.
134 citations
TL;DR: Measurements of the differential synthesis rate of ribosomal protein, alpha(r) (ribosomalprotein synthesis rate/total protein synthesis rate), indicate that ribosome protein synthesis is regulated directly or indirectly by availability of charged tRNA, and that the synthesis of ribsomal protein is subject to the influence of the rel gene control system.
Abstract: The regulation of the expression of ribosomal protein genes was examined during partial inhibition of tRNA aminoacylation in isogenic rel+ and rel- strains of E. coli B carrying a temperature-sensitive mutation in valyl-tRNA synthetase (EC 6.1.1.9) gene. Measurements of the differential synthesis rate of ribosomal protein, αr (ribosomal protein synthesis rate/total protein synthesis rate), indicate that ribosomal protein synthesis is regulated directly or indirectly by availability of charged tRNA, and that the synthesis of ribosomal protein, like the synthesis of rRNA, is subject to the influence of the rel gene control system.
108 citations
TL;DR: In this article, proteins from the 30 S ribosomal subunit of Escherichia coli were fractionated by column chromatography and individually incubated with 16 S RNA, and each of the RNA fragments was characterized by fingerprinting and positioned within the sequence of the 1600-nucleotide 16S RNA molecule.
103 citations
TL;DR: A diagonal polyacrylamide-dodecyl sulfate gel electrophoresis procedure is described, and the new crosslinked dimer, S4-S13, has been identified.
Abstract: A diagonal polyacrylamide-dodecyl sulfate gel electrophoresis procedure is described. Its utility is related to the use of the reagent methyl-4-mercaptobutyrimidate as a protein-protein crosslinking reagent. Crosslinking with this reagent occurs through the formation of intermolecular disulfide bonds. Oxidized proteins are separated in one dimension by electrophoresis under non-reducing conditions and in the second dimension under reducing conditions. All proteins except those derived from crosslinked species fall on a diagonal. Methods are described for the identification of the separated monomeric components of crosslinked species. The technique has been applied to the 30S ribosomal subunit of Escherichia coli, and the new crosslinked dimer, S4-S13, has been identified.
96 citations
TL;DR: Evidence is presented suggesting that the coordinate production of r- Proteins may result, in part, from a mechanism that degrades excess r-proteins that are not rapidly incorporated into ribosomal particles.
TL;DR: The results indicate that the ribosome efficiency increases by approximately 40% within the first few minutes following the shift-up of Escherichia coli strain B/r, implying that the protein to DNA ratio is constant for μ > 1.2 and equal to 4 ×10 8 to 5 × 10 8 amino acids/genome.
TL;DR: Poly(U)-dependent polyphenylalanine synthesis is completely dependent on the presence of ribosomal protein S1, and S1 is probably also required for the phage RNA-dependent binding of formylmethionyl-tRNA.
TL;DR: Application of the two-dimensional polyacrylamide gel electrophoretic procedure to the resolution of Escherichia coli ribosomal proteins is described and the positions in the electrophrogram of the individual proteins of the 30–S subunit have been determined.
Abstract: Application of the two-dimensional polyacrylamide gel electrophoretic procedure recently described by Mets and Bogorad [4] to the resolution of Escherichia coli ribosomal proteins is described. This procedure is fast (running time of 8–10 h) and highly sensitive (1–2 μg protein per spot) and it gave good resolution for the majority of the 55 proteins in E. coli ribosomes. Moreover, the method has permitted the resolution of a large number of high-molecular-weight proteins associated with ribosomes not exposed to high-salt washing. Using purified proteins, the positions in the electrophrogram of the individual proteins of the 30–S subunit have been determined.
TL;DR: The homology between the 30-S ribosomal protein S1 and interference factor i is investigated, which suggests that they have identical polypeptide chains.
Abstract: The homology between the 30-S ribosomal protein S1 and interference factor i has been investigated.
The two proteins are indistinguishable by gel electrophoresis under various conditions. Similar peptides are obtained by cleavage with cyanogen bromide. After labelling with N-ethylmaleimide, enzymatic digestion yields homologous labelled peptides. This homology is confirmed by immunological data.
Characteristic activities of the two proteins have been compared. Both bind polyuridylic acid and stimulate polypeptide synthesis on this template by S1-depleted ribosomes. Furthermore, S1 inhibits the translation of the coat protein cistron on MS2 RNA, while it has little effect on the overall translation of T4 mRNA, two characteristic properties of interference factor i.
S1 and factor i are thus physically and functionally indistinguishable, which suggests that they have identical polypeptide chains. Implications are discussed in terms of function.
TL;DR: The most satisfactory method for the extraction of protein from eukaryotic ribosomes is with 67% acetic acid in the presence of 33 mM MgCl2.
Abstract: Proteins were extracted from rat liver ribosomes and ribosomal subunits: with 67% acetic acid (in the presence of 3.3 mM, 33 mM, or 67 mM Mg) with 2 M LiCL in 4 M urea; with 0.25 N HCI; with 1% SDS; and after RNase digestion. The most efficient extraction and the best recovery were either with acetic acid in the presence of 33 mM or 67 mM Mg, or with LiCI-urea. Protein extracted with acetic acid, LiCi-urea, or with HCI had little or no contamination with RNA. The ribosomal proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis: the proteins extracted with acetic acid were the most soluble in the sample gel solution; their electrophoretograms displayed the maximum number of spots and the smallest number of derivatives or altered proteins. Preparations of protein extracted with SDS or RNase were relatively insoluble in the sample gel solution, and proteins extracted with HCI showed a large number of derivatives. All things considered, the most satisfactory method for the extraction of protein from eukaryotic ribosomes is with 67% acetic acid in the presence of 33 mM MgCl2.
TL;DR: A new relaxed mutant called rel C has been isolated from a rel A(+)/rel A(+) partial diploid strain and is unable to synthesize ppGpp or pppGpp in vivo in response to amino acid starvation or in vitro, but can synthesize these nucleotides in a shift-down.
Abstract: A new relaxed mutant called rel C has been isolated from a rel A+/rel A+ partial diploid strain. The rel C mutant is unable to synthesize ppGpp or pppGpp in vivo in response to amino acid starvation or in vitro, but can synthesize these nucleotides in a shift-down. Rel C maps near rif. Studies in vitro demonstrate the lesion to be probably in one of the 50S ribosomal proteins that can be removed by 1.0 M LiCl.
TL;DR: Questions are asked about the regulatory processes governing ribosome biosynthesis in prokaryotes: are all structural molecules of the ribosomes, RNA as well as proteins, regulated through one and the same basic mechanism?
Abstract: INTRODUCTION The ribosomes account for 40–50% of the cell mass of rapidly growing bacteria. It is therefore not surprising that the biosynthesis of ribosomes is a precisely regulated process requiring a strict coordination of the production of the 50–60 different molecules forming part of the ribosomal structures. At our present stage of ignorance, we have limited information about the regulatory processes governing ribosome biosynthesis in prokaryotes: Are all structural molecules of the ribosomes, RNA as well as proteins, regulated through one and the same basic mechanism? Is the biosynthesis of rRNA regulated at the level of initiation of transcription, at the level of the substrates, or does a breakdown mechanism of the nascent rRNA play a role? Is the biosynthesis of the ribosomal proteins regulated according to the Jacob-Monod model, or is there only an indirect control of the transcription frequency of the ribosomal protein operons through a common competition among all open promoters for the RNA polymerase molecules, as suggested by Maaloe (1969)? Other questions might also be asked: Does the regulatory mechanism primarily hit the ribosomal RNAs which in turn trigger the production of the ribosomal proteins? Inversely, are the ribosomal protein operons the prime target for the regulation, and do these proteins, or at least one of them, turn on the production of rRNAs? Are the ribosomal proteins protecting the nascent rRNAs against degradation, leaving excess rRNA chains at the prey of nucleases? The coordination between the rRNAs and the ribosomal proteins is a focal...
TL;DR: The model of active sites in E. coli ribosome illustrated in the figure is based on the presently available experimental results and should not be overinterpreted as an accurate topographical model.
TL;DR: This article summarizes and discusses the work related to ribosome assembly, concentrating mainly on the 30S subunit, including several recent developments in this area.
Abstract: INTRODUCTION The in vitro reconstitution of the bacterial 30S ribosomal subunit (Traub and Nomura 1968) followed by that of the 50S subunit (Nomura and Erdmann 1970; Fahnestock, Erdmann and Nomura 1973) has established that the information required for the assembly of this complex organelle is entirely contained in the structures of the RNA and protein components. The mechanism involved in this complex self-assembly process is now being elucidated, especially with respect to 30S ribosome assembly, by a variety of in vitro experiments. The in vitro assembly reaction, while differing in some details from in vivo assembly, very likely reflects the in vivo process in important ways. In addition, reconstitution techniques have facilitated the study of ribosome structure and structure-function relationships. A detailed review on the in vitro reconstitution of 50S subunits (Fahnestock, Held and Nomura 1972), as well as reviews on ribosome assembly in general (Nomura 1970Nomura 1973) have appeared previously. In this article, we summarize and discuss the work related to ribosome assembly, concentrating mainly on the 30S subunit, including several recent developments in this area. PARTIAL RECONSTITUTION OF RIBOSOMES The first step in the analysis of the functional role of ribosomal components was the development of a system for reconstituting ribosomes that had been partially disassembled (Staehelin and Meselson 1966; Hosokawa, Fujimura and Nomura 1966), Selective removal of a portion of the ribosomal protein (“split proteins”) was accomplished by centrifugation of the ribosomes in 5 M CsCl in the presence of 0.04 M Mg ++ . The functionally inactive nucleoprotein...
TL;DR: Treatment of both the 30-S and 50-S ribosomal subunits of Escherichia coli with dimethyl suberimidate, a cross-linking agent specific for protein amino groups, resulted in the formation of new protein species detectable by acrylamide gel electrophoresis in sodium dodecylsulfate.
Abstract: Treatment of both the 30-S and 50-S ribosomal subunits of Escherichia coli with dimethyl suberimidate, a cross-linking agent specific for protein amino groups, resulted in the formation of new protein species detectable by acrylamide gel electrophoresis in sodium dodecylsulfate. These new species were shown to contain two or more ribosomal proteins linked together and, after cleaving the cross-links with ammonia, these constituents were identified by two-dimensional electrophoresis.
TL;DR: Relative rates of ribosomal protein synthesis αr was determined by pulsechase labeling of cell protein followed by isolation of Ribosomes by electrophoresis of complete lysates on agarose gels, indicating that the free concentrations in the cell are not the same for all ribosome proteins.
Abstract: Relative rates of ribosomal protein synthesis αr was determined by pulsechase labeling of cell protein followed by isolation of ribosomes by electrophoresis of complete lysates on agarose gels. The agarose gel fractionation of lysates is described in detail. Cells growing in acetate, glucose and enriched glucose media had αr values of 0.09, 0.16, and 0.24, respectively. Estimates of the free pool of ribosomal protein were obtained from the kinetics of pulse-chase labeling of ribosomal particles (including precursor particles) and gave maximal values of 1.1, 2.1, and 3.1% of total ribosomal protein at the three different growth rates. The kinetics indicate that the free concentrations in the cell are not the same for all ribosomal proteins.
TL;DR: In this paper, it was shown that 50 S ribosomes of E. coli EA2 grown in the presence of ethionine are functionally abnormal and contain three methyl groups in a single N e,N e, N e -trimethyllysine residue and an equal number in another unidentified amino acid.
TL;DR: Proteins L7 and L12 from 50S ribosomal subunits of Escherichia coli are required for peptidechain termination and the binding of release factors to the ribosome appears to be the primary site of inhibition by thiostrepton.
Abstract: Proteins L7 and L12 from 50S ribosomal subunits of Escherichia coli are required for peptidechain termination. This termination process is inhibited by thiostrepton. Since both thiostrepton-treated ribosomes and those depleted of L7 and L12 have a markedly reduced ability to form release factor·UA[3H]A·ribosome complexes, the binding of release factors to the ribosome appears to be the primary site of inhibition.
TL;DR: In the 30S ribosomal subunit of Escherichia coli, two pairs of cross-linked proteins have been identified by several independent techniques; these are S7-S9 and S13-S19 as mentioned in this paper.
Abstract: Diimidoesters have been used to cross-link neighboring proteins in the 30S ribosomal subunit of Escherichia coli. Two pairs of cross-linked proteins have been identified by several independent techniques; these are S7-S9 and S13-S19. Since it has been shown earlier that these four proteins cooperate during ribosome assembly in vitro (Green and Kurland, 1973), their identification as near neighbors in the ribosome is quite suggestive. Thus, the data indicate that a cluster of proteins which interact with each other during assembly would be, in general, found in close proximity in the functional ribosome. A more detailed discussion of the arrangement of the proteins in the 30S subunit is presented.
TL;DR: A method to localize individual proteins on the surface of the ribosomal subunit was developed that uses specific immunoglobulins G against proteins under examination and points to the position of the protein the antibody was prepared against.
TL;DR: MS2 RNA binds at 0 degrees to 30S subunits from E. coli and to those of a Pseudomonas species, as judged by filtration on nitrocellulose membranes, and is competitive with such synthetic polynucleotides as poly(U) and poly(AUG).
Abstract: MS2 RNA binds at 0° to 30S subunits from E. coli and, to a smaller extent, to those of a Pseudomonas species, as judged by filtration on nitrocellulose membranes; this mRNA does not bind to 30S subunits from Bacillus brevis or Caulobacter crescentus. Binding does not depend on the presence of initiation factors; it is sensitive to aurintricarboxylic acid but insensitive to edeine and is competitive with such synthetic polynucleotides as poly(U) and poly(AUG). Complex formation can also be detected by electrophoresis on polyacrylamide-agarose gels. By this procedure, E. coli 30S subunits are separated into two major components. Only the more slowly moving component, which contains the ribosomal protein S1, interacts with the RNA.
TL;DR: The relative content in ribosomes of L7 and L12, the two forms of a protein in the 50S subunit specifically involved in GTP hydrolysis, is found to undergo a striking shift with the growth phase of E. coli, and the evidence suggests that the shift did not occur through modification of preexisting ribosome.
Abstract: The relative content in ribosomes of L7 and L12, the two forms of a protein in the 50S subunit specifically involved in GTP hydrolysis, is found to undergo a striking shift with the growth phase of E coli The content of L12 (nonacetylated form) increases during early logarithmic phase, becoming about 85% of the total before midlogarithmic phase Thereafter, L7 (N-acetylated form) content begins to increase, eventually becoming 75-80% in stationary phase The L7 + L12 content per ribosome, however, remained constant during this shift Our evidence suggests that the shift did not occur through modification of preexisting ribosomes The data further indicate that the E coli cell may contain more than one structurally distinct (with regard to L7 or L12 content) 50S subunit population