Te localization of eukaryotic initiation factor 3 (eIF-3) on native small ribosomal subunits has been established by electron microscopy through a comparison of native small ribosomal subunits with derived subunits and with native subunits stripped of eIF-3. Small subunits derived from reticulocyte ribosomes by the puromycin/KCI method are seen in electron micrographs as elongated particles, divided by a heavily stained partition into approximately one-third and twthird domains. Most particles (60-70%) observed in electron micrographs of native small subunit preparations resemble derived small subunits, but have an additional mass attached to one side, thus producing profiles with a three-lobed appearance. The mass measures approximately 160 X 100 X 60 A, and its particle weight is estimated to be about one-third to one-half tat of a 40S subunit. The site of attachment of the additional mass is located on a prominence extending from the central part of the small subunit and is separated by a cleft from the smaller third of the subunit. The remaining particles in preparations of native subunits resemble the profiles seen in electron micrographs of derived subunits. After removal of eIF-3 by treatment with high concentrations of salt, profiles observed in electron micrographs of washed, native subunits were indistinguishable from those of derived subunits. Since removal of eIF-3 coincided with removal of a mass of the correct molecular weight, subunits with the three-lobed appearance are identified as native small subunits carrying eIF-3.Preparations of native small ribosomal subunits from rabbit reticulocytes are able to carry out the initiation steps of protein synthesis when added to an in vitro system containing globin mRNA, derived large ribosomal subunits, and the components of the pH 5 enzyme fraction (1, 2). This ability is due to the presence of initiation factors, particularly to a large protein complex consisting of at least 10 polypeptides (3) which is attached to the native subunits. When isolated, the large protein complex corresponds in its sedimentation value and in the molecular weights of its components to eukarytoic initiation factor (eIF-3) (3-8). The large S value (15-17 S) and the estimated molecular weight (greater than 500,000) of the complex prompted us to attempt its localization by electron microscopy in preparations of native small ribosomal subunits. METHODSPreparation of Native Small Ribosomal Subunits. Native small ribosomal subunits were prepared from rabbit reticulocytes as described (1). In brief, blood from anemic rabbits (9) min at 20,000 X gay was recentrifuged twice: first for 45 min to remove polysomes and ribosome monomers and then for 5 hr at 105,000 X gay in a Ti6O rotor. The 5-hr sediment, containing the bulk of the native ribosomal subunits, was resuspended in solution A at a concentration of 120 A20 units/ml; 0.5-ml aliquots were layered onto 12 ml of 10%-40% sucrose density gradients containing 20 mM triethanolamine-HCI, pH 7.5/90 mM KCI/3 mM MgCl2/2 mM dithi...
The native small ribosomal subunit (S9) from rabbit reticulocytes which is able to initiate translation of globin mRNA in a cell-free system carries additional protein components. The latter can be separated from the subunit in a high salt sucrose gradient yielding a top-fraction (7) and a complex fraction (C), sedimenting at about 4 and 15 S, respectively. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed that fraction T contained four dominant polypeptides, while fraction C represents a large protein complex consisting of at least 10 polypeptides. SI' isolated from other sources showed similar patterns of their nonribosomal proteins.Reconstitution experiments revealed that fraction C is absolutely required for protein synthesis, while fraction T enhances protein synthesis only in the presence of C.The adherence of these protein factors to the subunit is not mediated by magnesium ions. Treatment of SO with EDTA and centrifugation in a magnesium-free sucrose gradient caused unfolding of the subunits and dissociation of several ribosomal proteins, but not of the factors. The unfolded ribosomal subunits sedimented as two distinct peaks. The more slowly sedimenting peak contained proteins of fraction T and the faster sedimenting one contained the 15S complex, indicating heterogeneity of the 9' population with respect to the factors attached to them.Initiation factors for protein synthesis in pro-and eukaryotes are in general found to be bound to ribosomes, from which they can be extracted by high concentrations of monovalent ions (1, 2). In eukaryotes it was recently established that the so-called native small ribosomal subunits (Sn), which comprise only a few percent of the total ribosomes, are able to initiate translation of mRNA in a reconstituted system containing derived large ribosomal subunits and pH 5 enzymes as the only other components (3, 4). Furthermore, it was reported that Sn contained several nonribosomal proteins (5) and that a salt extract from sn could stimulate globin mRNA translation in a cell-free protein synthesis system (6).These findings prompted us to identify the nonribosomal proteins found in Sn and to investigate their possible localization in other components of the ribosomal fraction, such as monosomes and polysomes, their mode of linkage to the small ribosomal subunit, their occurrence in a variety of cells, and their role in a reconstituted system. METHODSPreparation of Native Small Ribosomal Subunits (9'). Procedures were essentially as described (3). In brief: blood from anemic rabbits (7) was collected in the presence of cycloheximide. Further operations were performed in the cold (1-4°). Washed reticulocytes were lysed in 2 volumes of a solution of 10 mM Tris-HCl (pH 7.5)-10 mM KC1-1.5 mM MgCl2-2 mM dithiothreitol and homogenized with three strokes in a Potter-Elvehjem homogenizer. The postmitochondrial supernatant (10 min, 20,000 X gav) was centrifuged first for 30 min and the resulting supernatant was subsequently centrifuged again for 5 hr at 105,0...
A highly, active in vitro system for the translation of globin mRNA, resulting in more than 10 rounds of translation, is described. The reconstituted system consists of native small ribosomal subunits of rabbit reticulocytes (as a source of initiation factors as well as small ribosomal subunits), large subunits derived from rat liver polysomes by the puromycin-KCI procedure, and a pH 5 fraction obtained from a Krebs ascites cell high speed supernatant. In this system no differences were found between globin messenger ribonucleoprotein and globin mRNA.The special role which native ribosomal subunits play in the initiation of polypeptide synthesis in vitro was first recognized by Bishop (1). He showed that native ribosomal subunits present in reticulocyte lysates enhance the capacity of isolated reticulocyte polysomes to initiate globin chain synthesis. Subsequently it was shown that native subunits isolated from Escherichia coli were more active in viral RNA-dependent polypeptide synthesis than the 70S monomeric ribosomes, and that the high activity of the native ribosomal subunits was due to the localization of initiation factors on the small subunits (2, 3). In the case of eukaryotic native small subunits, they were found to be associated with additional nonribosomal protein, up to 7 X 105 daltons per subunit (4), Met-tRNAf (5) and mRNA (6). Recently, salt extracts from native small subunits of reticulocytes have been shown to result in initiation of polypeptide synthesis when added to a polypeptide-synthesizing system containing purified reticulocyte polysomes (7). In this paper we show that the native small ribosomal subunit isolated from rabbit reticulocytes can be used as a convenient source of initiation factors; together with large ribosomal subunits obtained by dissociation of rat liver polysomes by the puromycinzKCI procedure (8) and a pH 5 fraction obtained from a Krebs ascites cell high speed supernatant, they constitute a system that can translate globin messenger ribonucleoprotein (mRNP) more than 10 times. METHODS
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.