Regulation of synthesis of ribosomal protein in Neurospora crassa

In Neurospora crassa during a nutritional shift-down transition of growth, the synthesis of rRNA is for about 2 h largely inhibited and the rate of protein synthesis is only partially reduced (by about 25 %). During this period the relative rate of synthesis of individual ribosomal proteins, measured irrespectively of their incorporation into ribosomes, is reduced by 70–80%. The ribosomal proteins synthesized during the shift are stable. Thus, the synthesis of ribosomal proteins appears in N. crassa to be coordinately regulated with that of rRNA.     

Effects of two TMV strains on the synthesis and stability of chloroplast ribosomal RNA in tobacco leaves

Summary Multiplication of TMV-strains vulgare (light-green/dark-green mosaic symptoms) and flavum (severe yellow/green mosaic) had different effects on the ribosomal RNA of tobacco leaf chloroplasts. Vulgare inhibited chloroplast ribosomal RNA synthesis while having no effect on cytoplasmic ribosomal RNA synthesis (Fig. 2). Flavum inhibited chloroplast ribosomal RNA synthesis more severely than vulgare, and caused an earlier degradation of chloroplast ribosomal RNA than in control or vulgare-infected leaves (Fig. 1). Flavum also inhibited cytoplasmic ribosomal RNA synthesis. A connection between these differing effects on chloroplast ribosomal RNA metabolism and severity of visible symptoms is suggested, and discussed in relation to a possible influence on symptoms of denatured virus coat protein.Abbreviations TMV Tobacco Mosaic Virus - RNA Ribonucleic acid - DNA Deoxyribonucleic acid - m millions (in molecular weight values)     

Functional inactivation rates of the messenger RNA molecules coding for the individual ribosomal proteins in Escherichia coli.

Summary The rates of functional decay of messenger RNA coding for total soluble, total ribosomal and individual ribosomal proteins were measured in Escherichia coli strain AS-19, at 30^o. This was accomplished by blocking RNA synthesis with the inhibitor thiolutin and measuring residual protein synthesis at various times thereafter. The data obtained expressed as a decay constant (Hartwell and Magasanik, 1963) show that both total soluble and total ribosomal protein decay with similar rates (K ₂=0.64 and 0.61 respectively) which are slightly faster than the decay rate of -galactosidse (k ₂=0.43) under these conditions. All the individual ribosomal proteins appear to comprise a population of cistrons whose individual mRNA's decay with very similar rates with the possible exception of protein L3, whose mRNA appears consistently to decay very rapidly.Additional data on the stability of the total soluble and total ribosomal proteins during thiolutin treatment (that is, proteins synthesized in the absence of concommitant ribosomal RNA synthesis) fail to demonstrate any marked difference between these two protein populations. Examination of the stability of the individual ribosomal proteins however, reveals that some are degraded up to 35% in 15 min of thiolutin exposure, some to about 15% and some appear to be completely stable. In general, a degree of correlation exists between the stability of a given protein and the observed decay rate of its messenger RNA. This observation may explain in part the spread among the rates of mRNA decay. Nevertheless, we conclude that although degradation is occurring, it is not sufficient to alter the main conclusion that the rates of functional decay of mRNA cistrons coding for the ribosomal proteins are very similar.     

Changes in regulation of ribosome synthesis during different stages of the life cycle of Saccharomyces cerevisiae.

Summary Diploid strains of Saccharomyces cerevisiae, each homozygous for one of the temperature sensitive mutations rna2, rna4, rna6 or rna8, are temperature sensitive for ribosome synthesis during vegetative growth, but are not inhibited for ribosomal synthesis at the restrictive temperature under sporulation conditions. The continued ribosome biosynthesis at the restrictive temperature (34° C) during sporulation includes de novo synthesis of both ribosomal RNA and ribosomal proteins. This lack of inhibition of ribosome biosynthesis is found even when cells committed to complete sporulation are returned to vegetative growth medium. The ribosomes synthesized at 34° C are apparently functional, as they are found in polyribosomes. Although the rna mutants do not regulate ribosome synthesis during sporulation, all of these diploid strains fail to complete sporulation at 34° C. The cells are arrested after the second meiotic nuclear division but before ascus formation. The failure to complete sporulation at the restrictive temperature and the inhibition of ribosome biosynthesis during growth are caused by the same mutation, because revertants selected for temperature independent growth were also able to sporulate at 34° C.     

The phenotypic suppression of a mutation in the gene rplX for ribosomal protein L24 by mutations affecting the lon gene product for protease LA in Escherichia coli K12

Summary A suppressor mutation of a temperature-sensitive mutant of ribosomal protein L24 (rplX19) was mapped close to the lon gene by genetic analysis and was shown to affect protease LA. The degradation and the synthesis rates of individual ribosomal proteins were determined. Proteins L24, L14, L15 and L27 were found to be degraded faster in the original rplX19 mutant than in the rplX19 mutant containing the suppressor mutation. Other ribosomal proteins were either weakly or not at all degraded in both mutants. Temperature-sensitive growth was also suppressed by the overproduction of mutant protein L24 from a plasmid. Our results suggest that (1) either free ribosomal proteins or proteins bound to abortive assembly precursors are highly susceptible to the lon gene product and (2) the mutationally altered protein L24 can still function at the nonpermissive growth temperature of the mutant, if it is present in sufficient amounts.     

Loc1p is required for efficient assembly and nuclear export of the 60S ribosomal subunit

Loc1p is an exclusively nuclear dsRNA-binding protein that affects the asymmetric sorting of ASH1 mRNA to daughter cells in Saccharomyces cerevisiae. In addition to the role in cytoplasmic RNA localization, Loc1p is a constituent of pre-60S ribosomes. Cells devoid of Loc1p display a defect in the synthesis of 60S ribosomal subunits, resulting in “half-mer” polyribosomes. Previously, we reported that Loc1p is located throughout the entire nucleus; however, upon closer inspection we discovered that Loc1p is enriched in the nucleolus consistent with a role in 60S ribosome biogenesis. Given that Loc1p is an RNA-binding protein and presumably functions in the assembly of 60S ribosomal subunits, we investigated if Loc1p has a role in rRNA processing and nuclear export of 60S subunits. Analysis of pre-rRNA processing revealed that loc1Δ cells exhibit gross defects in 25S rRNA synthesis, specifically a delay in processing at sites A0, A1 and A2 in 35S pre-rRNA. Furthermore, loc1Δ cells exhibit nuclear export defects for 60S ribosomal subunits, again, consistent with a role for Loc1p in the assembly of 60S ribosomal subunits. It is attractive to hypothesize that the two phenotypes associated with loc1Δ cells, namely altered ASH1 mRNA localization and ribosome biogenesis, are not mutually exclusive, but that ribosome biogenesis directly impacts mRNA localization.     

Analysis of revertants of a ribosomal mutation in Podospora anserina: Evidence for new ribosomal mutations which confer hypersensitivity to paromomycin

This paper describes the analysis of cold-resistant revertants of a cold-sensitive mutant. Pm1-1 is a ribosomal mutation screened for its paromomycin resistance. Suppression of its cold sensitivity occurs with two kinds of external mutations localized in two different loci. One of them, PmB, is assumed to be a ribosomal gene. PmB mutations confer hypersensitivity to paromomycin in vivo as well as in vitro in a cell-free protein synthesis system.This work was supported by DGRST Grant MRM/P240 and NATO Grant 1637.     

Synthesis and characterization of amplified DNA in oocytes of the house cricket,Acheta domesticus (Orthoptera: Gryllidae)

At a time in the life cycle when a large proportion of the oocytes of Acheta incorporate ³H-thymidine into an extrachromosomal DNA body, synthesis of a satellite or minor band DNA, the density of which is greater than main band DNA, is readily detected. Synthesis of the satellite DNA is not detectable in tissues, the cells of which do not have a DNA body, or in ovaries in which synthesis of extrachromosomal DNA by the oocytes is completed. The DNA body contains the amplified genes which code for ribosomal RNA. However, less than 1 percent of the satellite DNA, all of which appears to be amplified in the oocyte, is complementary to ribosomal 18S and 28S RNA. In situ hybridization demonstrates that non-ribosomal elements, like the ribosomal elements of the satellite DNA, are localized in the DNA body.Abbreviations used rRNA ribosomal RNA, includes 18S and 28S RNA - rDNA gene sequences complementary to rRNA - cRNA complementary RNA synthesized in vitro     

Ribosomal proteins of Streptomyces aureofaciens producing tetracycline

Three different two-dimensional polyacrylamide gel electrophoretic systems were employed for identification of individual ribosomal proteins of Streptomyces aureofaciens. Proteins of small subunits were resolved into 21 spots. Larger ribosomal subunits contained 35 proteins. The separated ribosomal proteins from 50 S subunits were transferred on nitrocellulose membranes for immunochemical estimations. Antibodies developed against 50 S proteins of S. aureofaciens and Escherichia coli were used for identification of structural homologies between 50 S proteins of the two species. Results of the experiments indicate that about one half of the 50 S proteins of S. aureofaciens share common immunochemical determinants with corresponding proteins of 50 S subunits of E. coli. Evidence is presented that acidic ribosomal protein SL5 of large ribosomal subunits of S. aureofaciens can be assembled to E. coli P₀ cores lacking proteins L7/L12. Reconstitution of the P₀ cores with proteins SL5 or L7/L12 led to restoration of 78% activity in polyphenylalanine synthesis.     

Polyamine synthesis during lymphocyte activation : Induction of ornithine decarboxylase and S-adenosyl methionine decarboxylase

Lymphocyte stimulation by phytohaemagglutinin (PHA) is accompanied by marked increases in the activities of ornithine decarboxylase and S-adenosyl methionine decarboxylase, two key enzymes for the synthesis of polyamines. Both enzymes increase in a biphasic manner, with the rises in S-adenosyl methionine decarboxylase preceding the increases in ornithine decarboxylase. The initial rises precede the initiation of DNA synthesis, and seem to correlate with the increased rate of ribosomal RNA synthesis. Selective inhibition of ribosomal RNA synthesis inhibits the increases in the activity of both enzymes, especially ornithine decarboxylase, more than the increase in the overall rate of protein synthesis.Both enzymes are metabolically unstable and have half-lives of less than 1 h, although the half-life of ornithine decarboxylase depends on the amino acid concentration in the culture medium. While effects of PHA on the stability of the enzymes have not been ruled out, at least part of the PHA-dependent increases in activity are due to increased synthesis or activation of the enzymes. The synthesis of S-adenosyl-methionine decarboxylase declines rapidly after inhibition of RNA synthesis, but ornithine decarboxylase activity declines at about the same rate as protein synthesis as a whole.The activities of both enzymes also increase during lymphocyte stimulation by concanavalin A, lentil extract and staphylococcal filtrate.     

Telithromycin Inhibition of Protein Synthesis and 50S Ribosomal Subunit Formation in <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> Cells

The new ketolide antibiotic telithromycin (HMR3647) has been examined for inhibitory effects in cells of Streptococcus pneumoniae. The antibiotic caused a proportional decline in cell growth rate and viability with an IC₅₀ of 15 ng/ml. At a concentration of 7.5 ng/ml, protein synthesis in these cells was reduced by 50%. As seen in other organisms, this compound was also a very effective inhibitor of the formation of the 50S ribosomal subunit in growing cells. Pulse and chase labeling assays defined the reduced rate of 50S synthesis in antibiotic treated cells. At 7.5 ng/ml the rate was reduced to 50% of the control synthesis rate. An IC₅₀ of 15 ng/ml was found for the effect on this process. 30S ribosomal subunit formation was unaffected by the antibiotic. Inhibition of translation and 50S particle formation are equivalent targets for this antibiotic. The effects of telithromycin in S. pneumoniae are compared with those found in Staphylococcus aureus cells. Received: 29 October 2001 / Accepted: 1 February 2002     

Translational system of the hydrogen-oxidizing bacterium Alcaligenes eutrophus

Some structural and functional properties of ribosomes from the hydrogen-oxidizing bacterium Alcaligenes eutrophus were studied in order to investigate the background of expression of genetic information at the translational level. Ribosomal proteins from 30S subunits of A. eutrophus H16 were separated by two-dimensional gel electrophoresis into 21 spots, those from 50S subunits into 32 spots. While electrophoretic mobilities of several ribosomal proteins differed markedly from those of Escherichia coli, proteins sharing common immunological determinants with E. coli ribosomal proteins S1 and L7/L12 were found in A. eutrophus. Shifting from heterotrophic to autotrophic conditions of growth had no influence on the ribosomal protein pattern. Ribosomes of A. eutrophus had similar requirements for Mg²⁺ and poly(U) concentrations for optimum polyphenylalanine synthesis as those of E. coli. Protein synthesis elongation factors Tu from A. eutrophus and E. coli were immunologically similar. Efficiency of the A. eutrophus polyphenylalanine-synthesizing system was comparable to that of an analogous system derived from E. coli. This suggests that A. eutrophus could be employed for efficient expression of recombinant DNA.     

The pattern of protein and nucleic acid synthesis in germinating spores of Phycomyces blakesleeanus

Summary Synthesis of proteins, RNA and DNA is measured by incorporation of labelled precursors at different times during germination of Phycomyces spores.RNA and protein synthesis increases immediately after activation. DNa synthesis begins at a later stage (± 8 h) of germination when germ tubes are already present. Nuclear division occurs earlier in germination (±4–5 h) and is accompanied by a decrease in RNA synthesis. It can be concluded that at least most of the dormant spores are in the G2 phase of the cell cycle.Analysis of ribosomal RNA after pulse-chase labelling shows only three labelled compounds: a precursor molecule (2.25×10⁶ daltons) and the two mature ribosomal RNA compounds (1.4×10⁶ and 0.7×10⁶ daltons). This suggests that the two rRNAs are formed directly from the precursor molecule. Cycloheximide totally blocks the transformation of the ribosomal precursor molecule into mature rRNA.     

Structure of the terminal PCP domain of the non‐ribosomal peptide synthetase in teicoplanin biosynthesis

The biosynthesis of the glycopeptide antibiotics, of which teicoplanin and vancomycin are representative members, relies on the combination of non‐ribosomal peptide synthesis and modification of the peptide by cytochrome P450 (Oxy) enzymes while the peptide remains bound to the peptide synthesis machinery. We have structurally characterized the final peptidyl carrier protein domain of the teicoplanin non‐ribosomal peptide synthetase machinery: this domain is believed to mediate the interactions with tailoring Oxy enzymes in addition to its function as a shuttle for intermediates between multiple non‐ribosomal peptide synthetase domains. Using solution state NMR, we have determined structures of this PCP domain in two states, the apo and the post‐translationally modified holo state, both of which conform to a four‐helix bundle assembly. The structures exhibit the same general fold as the majority of known carrier protein structures, in spite of the complex biosynthetic role that PCP domains from the final non‐ribosomal peptide synthetase module must play in glycopeptide antibiotic biosynthesis. These structures thus support the hypothesis that it is subtle rearrangements, rather than dramatic conformational changes, which govern carrier protein interactions and selectivity during non‐ribosomal peptide synthesis. Proteins 2015; 83:711–721. © 2015 Wiley Periodicals, Inc.     

Genetic and biochemical studies of actidione sensitivity in Drosophila

A new mutant, act, a recessive gene carried on chromosome III of Drosophila, results in sensitivity to actidione, an inhibitor of protein synthesis. The mutant gene has no detectable effect except that it acts as a conditional lethal in the presence of actidione. The possibility that this gene encodes a ribosomal component was tested and eliminated using an in vitro system. Conditions are described for in vitro protein synthesis utilizing Drosophila microsomes and transfer RNA derived from Escherichia coli.This study was supported by USPHS Grant 1 R01 GM 15055.     

Eukaryotic ribosomal proteins stimulate Escherichia coli stringent factor to synthesize guanosine 5''-diphosphate, 3''-diphosphate (ppGpp) and guanosine 5''-triphosphate, 3''-diphosphate (ppGpp).

Summary When supplemented with Escherichia coli stringgent factor, 80S ribosomes from various sources failed to support guanosine tetra- and pentaphosphate ((p)ppGpp) synthesis. In contrast, ribosomal proteins from 80S, 60S or 40S particles (mouse embryos, rabbit reticulocytes) crossreacted with the E. coli stringent factor. Significant stimulation of (p)ppGpp synthesis was achieved proteins/ml. These observations may provide additional criteria to detect homologies between eukaryotic and prokaryotic ribosomal proteins.     

Chromosomal mutations causing resistance to tetracycline in Bacillus subtilis.

Summary We have isolated, after ethylmethanesulfonate mutagenesis, several chromosomal mutations causing resistance to tetracycline in Bacillus subtilis. These mutations fall into two classes, tetA and tetB. 30 S ribosomal protein S10 shows an altered mobility on two-dimensional acrylamide gels in cells bearing the former type of mutation. Ribosomes from these cells show elevated levels of resistance to tetracycline in vitro as measured by polyuridine dependent polyphenylalanine synthesis. The tetA locus maps adjacent to the tuf gene in the B. subtilis ribosomal protein gene cluster. Cells with the tetB mutation do not show any altered ribosomal protein, and their ribosomes are as sensitive, in vitro, to tetracycline as ribosomes isolated from wild type cells. The tetB mutation has been mapped proximal to cysA14.In partial fulfillment of the requirements for the doctoral degree by G.W. in the Department of Biology at the New York University Graduate School of Arts and Sciences     

Inhibition ofCandida utilis by cycloheximide and acetoxycycloheximide

Cycloheximide and acetoxycyloheximide were similar in their effect on the synthesis of RNA and protein by exponentially growing cells ofCandida utilis. Both antibiotics inhibited the synthesis of protein more than that of RNA. During inhibition there was preferential synthesis of ribosomal protein and some completed ribosomes were formed. However, the synthesis of ribosomal RNA was reduced more than that of transfer RNA. The actions of these drugs onCandida utilis are compared with the effects of other antibiotics whose primary effect on bacteria is to inhibit protein synthesis.