Initiation of mammalian protein synthesis

Schreier, Max H.; Erni, Bernhard; Staehelin, Theophil

doi:10.1016/0022-2836(77)90268-6

Cited by 299 publications

(114 citation statements)

References 18 publications

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“…The reaction mixture contained 25% lysate, 2 mM Mg-acetate, 150 mM K-acetate, 1 mM DTT, 1 mM ATP, 0.5 mM GTP, 4Opg of each amino acid except methionine, 50pg/ml rat liver tRNA, 50 mM spermine, 500 &i/ml [35S]methionine, crude initiation factors from a reticulocyte lysate [22] at 0.3 mg/ml final cont. and mRNA at 35 pg/ml.…”

Section: In Wtro Mrna Translationmentioning

confidence: 99%

The translation of the messenger for the poly(A)‐binding protein‐associated with translated mRNA is suppressed

et al. 1983

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In vivo protein synthesis in duck erythroblasts was compared to in vitro translation of polyribosomal and free cytoplasmic mRNA. The in vivo study showed the absence of de novo synthesis of the M r 73 000 poly(A)‐binding protein found associated with all polyribosomal mRNA. In vitro translation demonstrated that the mRNA for this protein is absent from the polyribosomal mRNA fraction but constitutes a medium frequency messenger among the repressed free mRNA. This result confirms the existence of a qualitative translational control in terminal differentiating duck erythroblasts leading eventually to the arrest of the protein synthesizing machinery.

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Section: In Wtro Mrna Translationmentioning

confidence: 99%

The translation of the messenger for the poly(A)‐binding protein‐associated with translated mRNA is suppressed

et al. 1983

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“…The eIF-2 was purified >90% from rabbit reticulocyte ribosomes according to the procedure of Schreier et al (14). The concentration of eIF-2 was determined by using the extinction coefficient E19m = 7.60(5); 1 jig of eIF-2 7 pmol, assuming a molecular weight of 140,000.…”

Section: Methodsmentioning

confidence: 99%

“…The gel slices were digested with 1 ml of 30% H202 at 370 overnight. After addition of 10 ml of toluene-based scintillation fluid (14), the samples were assayed for radioactivity in a Packard liquid scintillation counter (for 32p) or in a Packard gamma scintillation counter (for 125I). For double label counting (3H + 125I), the 3H radioactivity was corrected for the f radiation of 125I.…”

Section: Methodsmentioning

confidence: 99%

Binding and release of eukaryotic initiation factor eIF-2 and GTP during protein synthesis initiation.

Trachsel¹,

Staehelin²

1978

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

103

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The eukaryotic initiation factor eIF-2 forms a ternary corplex with Met-tRNAf and GTP. This complex binds to the 40S ribosomal subunit in the absence of mRNA and mRNA binding factors. Highly purified eIF-2 from rabbit reticulocytes was labeled with 12&I by using the Bolton-Hunter reagent or with [y-32P]ATP by using the heme-regulated translational inhibitor protein kinase. The labeled eIF-2 was bound, together with equimolar amounts of Met-tRNAf and GTP, to the 40S subunit. In the presence of mRNA, mRNA binding factors, and 60S ribosomal subunits (complete intiation assay), eIF-2 was released from the 40S initiation complex in the subunit joining reaction. GTP also was released in this step and probably was hydrolyzed in a reaction that is dependent upon eIF-5 and the 60S subunit. The function of phosphorylated eIF-2 in initiation of protein synthesis is discussed.

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“…mRNA initiation complex (p6) decreased during the 6-h incubaticn period, and that the amount of subunits containing initiation factors but probably not mRNA(p3) increased and that the fraction of free mRNP partk,les in the p2 region increased. factors have been isolated from the native 40-S subunit fraction [5,14,18] and characterised [15,16,18]. This complexity is reflected for example by the heterogeneity which most likely exists within the various classes of particles separated on CsCl gradients.…”

Section: Metaholic Shift-down Experimentsmentioning

confidence: 99%

Analysis of the Population of Native 40‐S Ribosomal Subunits in Mouse Plasmacytoma Cells Grown in Suspension Culture

Vangdal

Eikhom

1980

European Journal of Biochemistry

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The native ribosomal subunits of the mouse plasmacytoma cell line MPC-11 were fractionated by CsCl gradient centrifugation into seven classes of particles designated p l , p2, p3, p4, p5, p6 and p7, which possessed densities of approximately 1.38, 1.43, 1.46, 1.49, 1.50, 1.51 and 1.53 g/cm3 respectively. The RNA was analysed by oligo(dT)-cellulose chromatography and gel electrophoresis.In both well-fed and starved cells p3 was generally the dominating class of particles. During metabolic shift-up conditions, however, there was a great increase of p6 particles. Transfer of material containing rRNA and mRNA into this region of the CsCl gradient indicates the accumulation of an initiation complex containing mRNA.During metabolic shift-down conditions a decrease of p6 particles and an increase of p2 and p3 particles was observed. The p2 fraction was rich in mRNA and may contain considerable quantities of messenger ribonucleoprotein particles. A substantial fraction of newly synthesised mRNAcontaining material in starved cells banded in fact in the p2 region of the CsCl gradients. Circumstantial evidence indicates the existence of a transient 40-S initiation complex which bands in the p2-p3 region of the gradients. Double-labelling experiments demonstrated that radioactivity in 40-S subunits first appeared in precursor particles with a density of 1.49-1.50 g/cm3 and only later in p3 and p6 particles. p7 particles were detected in starved cells. They were slowly labelled and were presumably derived from old ribosomes following completion of the polysome cycle.We have previously demonstrated the existence of an excess of native ribosomal 40-S subunits (compared to native 60-S subunits) in the cytoplasm of rapidly growing transformed cells [1,2]. The excess was especially great in the plasmacytoma cell line MPC-11 following shift-up conditions [3]. In starved cultures, however, the ratio between the ribosomal subunits was approximately equimolar [l -31. It was of interest, therefore, to examine in greater detail the native 40-S subunit populations in starved and well-fed cells.It has been demonstrated that native 40-S ribosomal subunits can be fractionated on CsCl gradients into several subclasses depending upon the buoyant densities of the particles [4-71. One approach, therefore, to characterise the native 40-S ribosomal subunit population of starved and well-fed MPC-11 cells is to use CsCl gradient centrifugation in order to fractionate the subclasses. Old and newly Ahbreviutions. mRNP, messenger ribonucleoprotein; MettRNAf, the methionyl-tRNA species that initiates protein synthesis; eIF-3, eukaryotic initiation factor 3. synthesised rRNA and mRNA of the various subclasses can be analysed subsequently. Such an analysis is reported in this paper.The accumulation of a large pool of free native 40-S ribosomal subunits in MPC-11 cells following a metabolic shift-up makes these cells uniquely well suited to study certain aspects of initiation of protein synthesis in vivo and to follow the fate of newly synthesised 40-S r...

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Initiation of mammalian protein synthesis

Cited by 299 publications

References 18 publications

The translation of the messenger for the poly(A)‐binding protein‐associated with translated mRNA is suppressed

The translation of the messenger for the poly(A)‐binding protein‐associated with translated mRNA is suppressed

Binding and release of eukaryotic initiation factor eIF-2 and GTP during protein synthesis initiation.

Analysis of the Population of Native 40‐S Ribosomal Subunits in Mouse Plasmacytoma Cells Grown in Suspension Culture

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