Initiation of mammalian protein synthesis

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

doi:10.1016/0022-2836(77)90269-8

Cited by 324 publications

(111 citation statements)

References 18 publications

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“…By analyzing purified factors and proteins from initiation complexes side-by-side, after cross-linking, it was found that the latter polypeptides migrate in the same positions as three of the large subunits of eIF-3. This observation is consistent with both the facilitating effect of the cap on binding of mRNA to ribosomes (1) and eIF-3 promotion of 40S initiation complex formation with natural mRNAs (29,30). Differential interaction of the putative subunits of eIF-3 with the 5'-termini of different mRNAs and the ability of each mRNA to interact with more than one subunit might be related to the relative effi-…”

Section: '3supporting

confidence: 80%

“…Bergmann, J. Gordon and D. Baltimore). It is known that the majority of native 40S subunits contain eIF-3 (33,34), and the same factor binds specifically to 40S subunits in vitro (29,30). Consequently, it is tempting to suggest that in vivo a pre-initiation complex between mRNA and the 24,000 dalton capbinding polypeptide is directed to eIF-3-containing 40S ribosomal subunits at least partly by an interaction between the eIF-3 and the cap binding protein.…”

mentioning

confidence: 99%

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Interaction of a limited set of proteins with different mRNAs and protection of 5′-caps against pyrophosphatase digestion in initiation complexes

et al. 1979

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A variety of 5'-3H-methyl-labeled, oxidized viral mRNAs were used as probes for detecting in wheat germ initiation complexes proteins that interact with, and can be cross-linked to, the 5'-cap structure. A limited and reproducible set of specific proteins was obtained with the different mRNAs. The binding of these proteins to the 5'-end of mRNA apparently results in protection against nucleotide pyrophosphatase digestion of the cap even in initiation complexes in which the 5'-end is susceptible to pancreatic RNase digestion. Cross-linked proteins from mammalian initiation complexes comigrated with several of the subunits of similarly treated eIF-3. A model for cap binding protein interaction with mRNA cap during initiation of translation is suggested.

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Section: '3supporting

confidence: 80%

mentioning

confidence: 99%

Interaction of a limited set of proteins with different mRNAs and protection of 5′-caps against pyrophosphatase digestion in initiation complexes

et al. 1979

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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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“…in Materials and Methods ATP [26], as seen in Table 4. Both types of 80-S MettRNAy" complexes were analysed for stability to Met-tRNA:"' deacylase, as shown in Fig.9.…”

Section: Efpct Of' M E T -T R N a R E T Deacylase On Translational Inmentioning

confidence: 99%

Methionyl‐tRNA_f^Met Deacylase

Andersen¹

1979

European Journal of Biochemistry

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A methionyl‐tRNAfMet deacylase was found in ribosomal salt wash from cultured human cells of the HeLa line. This enzyme was purified by the use of DEAE‐cellulose, ammonium sulfate precipitation, gel filtration and isoelectric focusing, and appears to be a protein with a native molecular weight of 80000, which consists of two 40000‐Mr subunits. The mechanism of the Met‐tRNAfMet deacylase is shown to involve end‐product inhibition by the deacylated form of MettRNAfMet. The methionyl‐tRNAfMet deacylase is rather specific for Met‐tRNAfMet as opposed to Met‐tRNAmMet, has a KCl optimum of 85 mM, is inhibited by MgCl2 and is inhibited by GTP and NAD+ at physiological concentration. 40‐S and 60‐S subunits inhibit the enzyme, possibly by binding to it. The stability of translational initiation complexes, containing methionyl‐tRNAfMet, was investigated in the presence of the enzyme. Purified ternary complex was slowly broken down by the enzyme, while the 40‐S‐subunit · Met‐tRNAfMet complex was stable in the presence of enzyme. The 80‐S complex formed with A‐U‐G trinucleotide as the message molecule was broken down, whereas the 80‐S complex formed with globin mRNA was stable in the presence of the enzyme. The physiological role of this enzyme is unclear, but it might act to regulate initiation by deacylating Met‐tRNAfMet.

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Initiation of mammalian protein synthesis

Cited by 324 publications

References 18 publications

Interaction of a limited set of proteins with different mRNAs and protection of 5′-caps against pyrophosphatase digestion in initiation complexes

Interaction of a limited set of proteins with different mRNAs and protection of 5′-caps against pyrophosphatase digestion in initiation complexes

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

Methionyl‐tRNA_f^Met Deacylase

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Initiation of mammalian protein synthesis

Cited by 324 publications

References 18 publications

Interaction of a limited set of proteins with different mRNAs and protection of 5′-caps against pyrophosphatase digestion in initiation complexes

Interaction of a limited set of proteins with different mRNAs and protection of 5′-caps against pyrophosphatase digestion in initiation complexes

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

Methionyl‐tRNAfMet Deacylase

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Product

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About

Methionyl‐tRNA_f^Met Deacylase