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W K Roberts

Publications and source records attributed to W K Roberts.

8 recordsLinked to original sources
PubMed1979

Purification and properties of a translation inhibitor from wheat germ.

A translation inhibitor from wheat germ has been purified more than 400-fold to apparent homogeneity. The inhibitor is a basic protein with a molecular weight of 30 000. This protein effectively blocks protein synthesis in animal cell-free extracts but does not affect protein synthesis in intact cells. Inhibition occurs at a ribosome to inhibitor molar ratio of 100:1, indicating an enzymic mechanism of action. The wheat germ protein inhibits the translation of endogenous mRNA, exogenous mRNA, and poly(uridylic acid) at a step in polypeptide chain elongation and without breakdown of the polysomes. Neither the aminoacylation reaction nor mRNA degradation is affected by the inhibitor. An interesting feature of the inhibition reaction is that it requires, in addition to the wheat germ inhibitor, both ATP and tRNA. The function of these two compounds in the inhibition is presently unknown since neither the hydrolysis of the beta,gamma-pyrophosphate bond of ATP nor a modification of the tRNA can be demonstrated during the reaction.

Kinetics↗
PubMed1977

An ATP-dependent inhibition of protein synthesis in ascites cell extracts by wheat germ protein.

A high speed supernatant fraction from wheat germ was shown to be a very effective inhibitor of protein synthesis in a cell-free protein synthesizing system from Ehrlich ascites cells. Low concentrations of the extract were equally effective in inhibiting the translation of EMC viral RNA, ascites cell mRNA (exogenous) and endogenous mRNA. The kinetics of inhibition in the presence and absence of pactamycin, as well as the observed inhibition of polyphenylalanine synthesis, indicate that the wheat germ inhibitor acts at the level of elongation. Preincubation of the ascites system with the wheat germ inhibitor in the presence and absence of ATP showed that ATP was required for the development of the inhibition. The inhibitor was partially purified and appears to be a basic protein with a molecular weight of 30 000--40 000. These results are discussed with respect to the hypothesis that this may be another example of a protein kinase-induced inhibition of protein synthesis.

Adenosine Triphosphate↗
PubMed1977

Encephalomyocarditis virus RNA. II. Polyadenylic acid requirement for efficient translation.

Differentially polyadenylated subpopulatons of encephalomyocarditis (EMC) viral RNA were isolated by affinity chromatography on oligodeoxythymidylic acid-cellulose. Translation of these RNA fractions in several in vitro protein-synthesizing systems, isolated from Ehrlich ascites tumor cells, demonstrated that poly(A)+EMC viral RNA was translated two to three times more efficiently than poly(A)-EMC viral RNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the polypetides synthesized by the in vitro system in response to the different RNAs showed no detectable differences in the size or relative amount- of the translational products. mRNA saturation curves indicated that the in vitro systems were stimulated maximally by equivalent amounts of RNA, wheter it be poly(A)-or poly(A)+ EMC viral RNA. Time course experiments showed that the differences in translatability were more pronounced late in the reaction when reinitiation was required, and that by eliminating reinitiation with high salt the apparent effect of poly(A) on translation was diminished. Together, these results suggest that poly(A) may be required for efficient initiation and reinitiation of protein synthesis in the cell-free systems. This interpretation is discussed relative to earlier data.

Animals↗
PubMed1976

Limitation of reticulocyte transfer RNA in the translation of heterologous messenger RNAs.

The effect of various tRNAs on protein synthesis was investigated using a tRNA-dependent cell-free system from Ehrlich ascites cells. Ascites cell tRNA and rabbit liver tRNA were found to promote efficient translation of globin mRNA, oviduct mRNA, and encephalomycarditis (EMC) viral RNA. In contrast, reticulocyte tRNA participated efficiently only in the translation of globin mRNA; the translation of oviduct mRNA AND EMC viral RNA in the presence of reticulocyte tRNA resulted in the synthesis of relatively few large mature proteins and the accumulation of discrete, smaller polypeptides. These results suggest that isoaccepting tRNA species required for the synthesis of ovalbumin and EMC viral protein (but not hemoglobin) are probably functionally absent in reticulocyte tRNA, causing a premature, nonrandom termination of synthesis of these proteins. This provides preliminary evidence that variations in tRNA populations, frequently observed between different cell types, are large enough to define and perhaps regulate the proteins that the cell is capable of synthesizing.

Animals↗
PubMed1976

Interferon-induced inhibition of protein synthesis in L-cell extracts: an ATP-dependent step in the activation of an inhibitor by double-stranded RNA.

The translation of encephalomyocarditis virion RNA in extracts from interferon-treated L-cells is inhibited by the addition of double-stranded RNA (dsRNA) at 400 ng/ml. A similar inhibition in response to dsRNA is seen in control cell extracts supplemented with small amounts of a postribosomal supernatant fraction from interferon-treated cells (interferon cell sap): Neither interferon cell sap nor dsRNA alone is inhibitory in control systems. The inhibition is much reduced if translation is carried out at low ATP concentrations.Conversely, the inhibitory capacity of the interferon cell sap is increased 100-fold if it is preincubated with dsRNA and ATP prior to its addition to the protein-synthesizing system. After this preincubation all detectable dsRNA can be removed without any diminution of the inhibitory activity of the cell sap. These results are compatible with a two-step model for the inhibition in which a pre-inhibitor is activated by dsRNA, the activated inhibitor then interacting with the protein synthesis system to inhibit translation.

Adenosine Triphosphate↗
PubMed1976

Encephalomyocarditis virus RNA: variations in polyadenylic acid content and biological activity.

Encephalomyocarditis (EMC) viral RNA was isolated from purified virus grown in Ehrlich ascites tumor cells. The viral RNA was found to contain polyadenylic acid [poly(A)] regions that were very heterogeneous in length. Chromatography of the EMC viral RNA on oligo(dT)-cellulose columns separated the RNA into three distinct fractions (peaks 1 to 3). Approximately 20% of the EMC viral RNA appeared as peak 1, 40% as peak 2, and 40% as peak 3. The RNA in each fraction appeared to be intact as shown by co-sedimentation with 35S unfractionated EMC viral RNA in SDS-sucrose density gradients. Approximately 95 to 100% of peaks 1 and 3, and 60 to 70% of peak 2, reappeared at the same elution position after rechromatography on oligo(dT)-cellulose. The RNA in peak 1 contained poly(A) with an average length of 16 nucleotides, peak 2 contained poly(A) with an average of 26 nucleotides, and peak 3 contained an average of 74 nucleotides in its poly(A) region. The distribution in the three fractions, as well as the average length of the poly(A) moieties, was relatively unaffected by changes in the cell suspension medium used during infection. Finally, each of the three viral RNA fractions was assayed for biological activity using an infectious RNA assay on L-cell monolayers. Infectivity of the viral RNA was found to increase with poly(A) length, with peak 3 viral RNA being approximately 10 times more infectious than peak 1 viral RNA.

Encephalomyocarditis virus↗
PubMed1975

A transfer RNA-dependent protein synthesizing system from Ehrlich ascites extracts.

An homologous cell-free system from Ehrlich ascites cells was made tRNA dependent by passing both the ribosomes and the ammonium sulfate fraction through DEAE-cellulose columns in 0.30 M KCl. Protein synthesis was routinely stimulated 20-50 fold by the addition of 100 mug/ml of ascites cell tRNA in the presence of oviduct mRNA, ascites mRNA, or encephalomyocarditis RNA.

Animals↗