Hepatitis C virus NS5B RNA replicase specifically binds ribosomes

Hepatitis C virus (HCV) non-structural protein 5B (NS5B) is an RNA replicase. We expressed full-length NS5B (591 amino acid residues) in Escherichia coli as a fusion protein with maltose binding protein (MBP-NS5B). MBP-NS5B was recovered in the soluble fraction after centrifugation at 40,000 x g and affinity-purified with amylose resin. The purified MBP-NS5B had a high-level of poly (A), oligo (U)-dependent UMP incorporation with a Km of 2 microM for UTP. Surprisingly, the enzymatically active MBP-NS5B was sedimented by ultracentrifugation at 160,000 x g. The pellet contained 16S and 23S ribosomal RNAs, suggesting that ribosomes were associated with MBP-NS5B. Ribosomes and MBP-NS5B were subsequently co-purified on amylose resin. Deletion study revealed that either the N-terminal (amino acid residues 1-107) or the C-terminal (amino acid residues 498-591) region of NS5B were sufficient for this association with ribosomes. We further found that NS5B also bound with human ribosomes. Our results implicate a novel mechanism of coupling between replication and translation of the viral genome in the life cycle of HCV.     

Arabidopsis Coordinates the Diurnal Regulation of Carbon Allocation and Growth across a Wide Range of Photoperiods

In short photoperiods, plants accumulate starch more rapidly in the light and degrade it more slowly at night, ensuring that their starch reserves last until dawn. To investigate the accompanying changes in the timing of growth, Arabidopsis was grown in a range of photoperiods and analyzed for rosette biomass, photosynthesis, respiration, ribosome abundance, polysome loading, starch, and over 40 metabolites at dawn and dusk. The data set was used to model growth rates in the daytime and night, and to identify metabolites that correlate with growth. Modeled growth rates and polysome loading were high in the daytime and at night in long photoperiods, but decreased at night in short photoperiods. Ribosome abundance was similar in all photoperiods. It is discussed how the amount of starch accumulated in the light period, the length of the night, and maintenance costs interact to constrain growth at night in short photoperiods, and alter the strategy for optimizing ribosome use. Significant correlations were found in the day- time and the night between growth rates and the levels of the sugar-signal trehalose 6-phosphate and the amino acid biosynthesis intermediate shikimate, identifying these metabolites as hubs in a network that coordinates growth with diurnal changes in the carbon supply.     

Changes in gene expression of Scots pine buds during the winter and under experimentally altered light and temperature conditions

Vegetative buds of Scots pine ( Pinus sylvestris L.) were collected during the apparently dormant phase in January and February and at the beginning of the growth phase in May. Wintering Scots pine plants were placed in climate chambers in which either the daily photoperiod or the temperature simulated the situation in early spring, whereas the other conditions were characteristic of midwinter.
The amount of total ribosome populations and their in vitro translation capacities were independent of the height of the tree or place of collection, but both were dependent on the season; the amount of ribosomes per fresh weight of buds was lower in spring than in winter, whereas the translation capacity in spring exceeded that in winter. Poly(A)⁺ RNA was purified from the ribosomes and translated in vitro and the translation products were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The polypeptide patterns proved that changes in gene expression can occur in plants growing outdoors even during the season of severe cold. In the climate chamber experiments, lengthening of the daily photoperiod increased the in vitro translation capacity of the buds within 7 days even at temperatures below 0°C, whereas a rise in temperature seemed to cause a more transient stress effect. Both treatments induced alterations in the pattern of in vitro polypeptide synthesis. It is suggested that while improvements in both light and temperature hastened the development of the buds under experimental growth conditions, the lengthening of the day may be the factor which induces a change in wintertime metabolism under natural conditions.     

“In situ” translation: Use of the cytoskeletal framework to direct cell-free protein synthesis

Summary We have developed a novel, “in situ” translation system derived from cultured cells that are subject to mild detergent extraction. By using a low concentration of nonionic detergent to gently permeabilize cells while they remain adherent to a substrate, cytoskeletal frameworks are obtained that are devoid of membraneous barriers yet retain much the same topological arrangement of mRNA, ribosomes and cytostructure that exists “in vivo”. Data indicate that when these cytoskeletal frameworks are supported by a ribosome-depleted, nuclease-treated, reticulocyte lysate supernatant, they are capable of resuming translation of their attached polysomes for at least 40 minutes. Emulsion autoradiography of ongoing protein synthesis demonstrates that protein synthetic activity is ubiquitous throughout the population of extracted cells, and not confined to a less well-extracted subset. Computer-assisted, two-dimensional gel analysis reveals that the pattern of proteins produced by such extracted cells is approximately 70% coincident with that produced by unextracted cells, including proteins of molecular weight as great as 200 kilodaltons. Furthermore, a continued increase in intensity of almost all proteins during the first 40 minutes of translation suggests that translational re-initiation, in addition to polysome run-off, is also taking place. Collectively, these findings indicate that much of the translational machinery remains both intact and competant in this cytoskeletal-based translation system. As such, this system should prove extremely useful in identifying molecular factors operant during certain types of translation control and in further examining the role played by the cytoskeleton in regulating gene expression. This work was supported by grants from the American Cancer Society (#NP-683) and from the University of Connecticut Health Center Research Foundation.     

A nuclear mutation conferring thiostrepton resistance in Chlamydomonas reinhardtii affects a chloroplast ribosomal protein related to Escherichia coli ribosomal protein L11

We have isolated a nuclear mutant (tsp-1) of Chlamydomonas reinhardtii which is resistant to thiostrepton, an antibiotic that blocks bacterial protein synthesis. The tsp-1 mutant grows slowly in the presence or absence of thiostrepton, and its chloroplast ribosomes, although resistant to the drug, are less active than chloroplast ribosomes from the wild type. Chloroplast ribosomal protein L-23 was not detected on stained gels or immunoblots of total large subunit proteins from tsp-1 probed with antibody to the wild-type L-23 protein from C. reinhardtii. Immunoprecipitation of proteins from pulse-labeled cells showed that tsp-1 synthesizes small amounts of L-23 and that the mutant protein is stable during a 90 min chase. Therefore the tsp-1 phenotype is best explained by assuming that the mutant protein synthesized is unable to assemble into the large subunit of the chloroplast ribosome and hence is degraded over time. L-23 antibodies cross-react with Escherichia coli r-protein L11, which is known to be a component of the GTPase center of the 50S ribosomal subunit. Thiostrepton-resistant mutants of Bacillus megaterium and B. subtilis lack L11, show reduced ribosome activity, and have slow growth rates. Similarities between the thiostreptonresistant mutants of bacteria and C. reinhardtii and the immunological relatedness of Chlamydomonas L-23 to E. coli L11 suggest that L-23 is functionally homologous to the bacterial r-protein L11.     

The Ribonucleic Acids of Crithidia fasciculata*

SYNOPSIS. Crithidia fasciculata ribosomes were found to be 80S and to dissociate into 58 and 41S subunits; on 5 to 50% sucrose gradients, rRNA was separated into 25, 18, and 5S components. The molecular sizes of the heavier rRNA species, estimated by polyacrylamide gel electrophoresis were 1.24 and 0.84 M (×10⁶ daltons). The 25S RNA has a tendency to interact with the 18S RNA to give a complex that is difficult to separate by sucrose gradient centrifugation. The 25S RNA is also unstable and dissociates into 0.73 and 0.57 M components. The 18S RNA has molecular size (0.84 M) higher than the 0.7 M reported for most eukaryotes, but similar to that of Euglena and Amoeba. Ribosomal RNA hybridized 0.29% of the nuclear DNA. Mitochondrial RNA, extracted by a rapid procedure was resolved into 16 and 5S components in sucrose gradients.     

Ribosome crystallization in nuclei of chick embryos

Ribosome crystallization within nuclei has been studied in chick embryos with procedures which increase its frequency by various orders of magnitude as compared to previous findings. The extrusion of ribosome microcrystals from nuclei is reported for the first time, and a model for the transfer of ribosomes from nucleus to cytoplasm is proposed.     

The accessibility of antigenic determinants of ribosomal protein s4 in situ

Antibodies to Escherichia coli ribosomal protein S4 react with S4 in subribosomal particles, eg, the complex of 16S RNA with S4, S7, S8, S15, S16, S17, and S19 and the RI* reconstitution intermediate, but they do not react with intact 30S subunits. Antibodies were isolated by three different methods from antisera obtained during the immunization of eight rabbits. Some of these antibody preparations, which contained contaminant antibodies directed against other ribosomal proteins, reacted with subunits, but this reaction was not affected by removal of the anti-S4 antibody population. Other antibody preparations did not react with subunits. It is concluded that the antigenic determinants of S4 are accessible in some protein deficient subribosomal particles but not in intact 30S subunits.