Myosin synthesis and specificity of eukaryotic initiation factors

Rourke, A. W.; Heywood, Stuart M.

doi:10.1021/bi00761a010

Cited by 86 publications

(30 citation statements)

References 12 publications

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“…Twentyfive one hundredth nmol each of 20 amino acids containing 8 jACi of 3H-labeled amino-acid mixture (New England Nuclear Corp.) was added to each incubation mixture. Where indicated, 2 ,g of tcRNA and 15 ug of poly(A)-containing mRNA from muscle were added to the cell-free, amino-acid incorporating systems.…”

Section: Methodsmentioning

confidence: 99%

“…In order to remove most of the radioactive globin synthesized during the incubation, we dialyzed the cell-free systems against 55% (NH4)2SO4 (pH 7.0) at 50 after completion of the incubation period. The precipitated radioactive proteins were subsequently analyzed by Na dodecyl sulfate-acrylamide gel electrophoresis on 7% gels, as described for myosin (2). The radioactivity of the proteins synthesized was determined by liquid scintillation counting after the acrylamide gels were sliced (2).…”

Section: Methodsmentioning

confidence: 99%

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Two classes of translational control RNA: their role in the regulation of protein synthesis.

Bester¹,

Kennedy²,

Heywood³

1975

Proc. Natl. Acad. Sci. U.S.A.

104

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Two classes of translational control RNA (tcRNA) have been isolated from embryonic chick muscle. One of these classes, the tcRNA isolated from messenger ribonucleoprotein particles (mRNP-tcRNA), is effective in inhibiting the translation of mRNP-mRNA while having little if any effect on polysomal mRNA. The other class, polysome-tcRNA, has no effect on mRNP-mRNA while it stimulates the translation of polysomal mRNA. Translational control RNA (tcRNA) has been isolated from initiation factor preparations and has been demonstrated to specifically inhibit the translation of heterologous mRNAs (3). The inhibition by muscle tcRNA results from the inability of the globin mRNA to bind to ribosomes during the formation of the initiation complex when tcRNA is added to the cell-free system (6). Due to the specific nature of this inhibition, it is likely that the tcRNA interacts directly with mRNA. However, the involvement of initiation factors in this tcRNA-mediated inhibition cannot be excluded.In order to determine if tcRNA is effective in the regulation of protein synthesis within the cell from which it is derived, we have isolated mRNA from both polysomes and messenger ribonucleoprotein particles (mRNPs) of embryonic chick muscles and tested the ability of muscle tcRNA to inhibit the translation of these mRNAs. We report here that tcRNA may be obtained from either the polysomal or mRNP sources. Both tcRNA preparations inhibit globin synthesis, while only the tcRNA obtained from mRNPs is effective in inhibiting the Abbreviations: tcRNA, translational control RNA; mINP, messenger ribonucleoprotein particles. * Permanent address: Tygerberg Hospital, Capetown, South Africa. 1523translation of muscle mRNAs; and, in this case, only the translation of mRNP-mRNA is inhibited. It therefore is likely that at least two classes of tcRNA are present in muscle. It was of particular interest that the tcRNA effective in inhibiting muscle mRNP-mRNA translation contains a poly(U) tract that is absent in the polysomal class of tcRNA. From these results, a model is proposed to explain the manner by which tcRNA specifically regulates the utilization of cytoplasmic mRNAs. MATERIALS AND METHODSThe preparation of rabbit reticulocyte lysates and the conditions for cell-free protein synthesis were as described (3) except that 50 AM hemin and 15 jAg of muscle initiation factor 3 (IF3) were present in the reaction mixture, which had a final volume of 0.1 ml. Incubations were for 30 min at 300. Twentyfive one hundredth nmol each of 20 amino acids containing 8 jACi of 3H-labeled amino-acid mixture (New England Nuclear Corp.) was added to each incubation mixture. Where indicated, 2 ,g of tcRNA and 15 ug of poly(A)-containing mRNA from muscle were added to the cell-free, amino-acid incorporating systems.The mRNA and tcRNA preparations were prepared from 14-day embryonic chick leg muscle in the following manner. Approximately 45-50 g of muscle was gently homogenized in 0.25 M KC1, 5 MM M\gCI2, 20 mM Tris-HCl (pH 7.4) as described (7). The 10,00...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Two classes of translational control RNA: their role in the regulation of protein synthesis.

Bester¹,

Kennedy²,

Heywood³

1975

Proc. Natl. Acad. Sci. U.S.A.

104

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“…Previous results have suggested that red muscle may contain a number of message specific factors (1). In order to separate these factors into fractions specific for either myosin or myoglobin, the IF-3 fraction from red muscle was chromatographed on phosphocellulose.…”

Section: Resultsmentioning

confidence: 99%

“…Both highly purified myosin and myoglobin as well as their mRNAs were prepared as described (1,2 arations contain other RNA species and, therefore, the amount of individual mRNA present is not known. Initiation factors (IF-1, IF-2, and IF-3) were prepared from 100 to 150 g of 19-day embryonic chick red muscle.…”

Section: Methodsmentioning

confidence: 99%

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Separation of Specific Initiation Factors Involved in the Translation of Myosin and Myoglobin Messenger RNAs and the Isolation of a New RNA Involved in Translation

Heywood

Kennedy

Bester

1974

Proc. Natl. Acad. Sci. U.S.A.

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Two messenger specific factors, phosphocellulose fractions 3 and 4, have been isolated from the initiation factor 3 fraction of red muscle initiation factors by chromatography on phosphocellulose. When added to a reticulocyte cell-free system containing both myoglobin and myosin mRNAs, phosphocellulose fraction 3 is found to specifically stimulate the synthesis of myoglobin while phosphocellulose fraction 4 is found to specifically stimulate the synthesis of myosin. In addition, a new RNA, isolated from the initiation factor 3 fraction, is shown to specifically inhibit the translation of heterologous mRNAs.Myosin and myoglobin mRNA have been translated in heterologous cell-free systems (1, 2). In these systems the mRNAs to be tested have competed with endogenous mRNAs for the various constituents of the cellular apparatus involved in protein synthesis. Under these conditions, the addition of specific initiation factor 3 (IF-3) fractions has been required to make this competition more favorable and allow for more efficient synthesis of these proteins. Red muscle cells contain specific factors for the translation of both myosin and myoglobin, whereas white muscle cells lack the specific initiation factor for myoglobin synthesis (2). These results have led us to suggest that muscle cells contain a multiplicity of specific initiation factors. Recently, several reports utilizing globin and viral mRNAs have also suggested that messenger specific initiation factors are found in eukaryotic cells (3,4). In addition to specific protein factors involved in the translational control of protein synthesis, a number of investigators have raised the possibility that an RNA molecule may also be involved in translational control by directly effecting the initiation of protein synthesis (5-8).We report here the separation of myosin and myoglobin specific factor activity by chromatography of red-muscle IF-3 on phosphocellulose. Multiple protein fractions are obtained, and two of these fractions are found to have different specificities in translating myosin and myoglobin mRNA in a reticulocyte cell-free system. In addition, a new RNA species, isolated from IF-3 fraction, is shown to specifically inhibit the translation of heterologous mRNAs. This RNA has been termed translation control RNA (tcRNA).

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The Molecular Defect in Thalassaemia

Clegg

1976

Ciba Foundation Symposium 37 ‐ Congenital Disorders of Erythropoiesis

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Myosin synthesis and specificity of eukaryotic initiation factors

Cited by 86 publications

References 12 publications

Two classes of translational control RNA: their role in the regulation of protein synthesis.

Two classes of translational control RNA: their role in the regulation of protein synthesis.

Separation of Specific Initiation Factors Involved in the Translation of Myosin and Myoglobin Messenger RNAs and the Isolation of a New RNA Involved in Translation

The Molecular Defect in Thalassaemia

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