Polypeptide Chain Initiation and Stepwise Elongation with Artemia Ribosomes and Factors

Abstract

The supernatant initiation factor from Artemia salina embryos promotes, besides the AUGdependent binding of fMet-tRNAf, the poly(U)-dependent binding of N-acetylPhe-tRNA to 40S ribosomal subunits; the bound N-acylaminoacyl-tRNA reacts directly with puromycin upon addition of 60S subunits. Both the binding reaction and the synthesis of N-acylaminoacyl-puromycin occur in the absence of GTP or other ribonucleoside triphosphates. To a smaller extent, the factor also mediates the 40S ribosomal binding of Met-tRNAf and PhetRNA; in this case, the bound aminoacyl-tRNA is less reactive with puromycin. After the poly(U)-and supernatant factor-dependent binding of N-acetylPhe-tRNA to 40S subunits at low Mg2+ concentration, binding of a second aminoacyl-tRNA (Phe-tRNA), with ensuing formation of the first peptide bond, is dependent upon the addition of the 60S subunit, elongation factor EF-1, and GTP. Further growth of the polypeptide chain requires translocation and is, therefore, dependent upon the addition of elongation factor EF-2. As with the Escherichia coli system, once requirements for translation of the third codon have been met, no further additions are necessary for elongation of a peptide chain.In previous papers (1, 2), we reported on an initiation factor from postribosomal supernatants of embryos of the brine shrimp, Artemia salina, and its relation to factors from ratliver supernatant (3-6) and rabbit-reticulocyte ribosomal washes (7) described by other investigators. The Artemia factor and the Escherichia coli initiation factor IF-2 are not interchangeable (2) but, as previously noted (1), the reaction promoted by Artemia factor, namely the AUG-dependent binding of fMet-tRNAf to eukaryotic 40S ribosomal subunits, shares certain properties with the analogous reaction catalyzed by IF-2 in bacterial systems: (a) it occurs on the small ribosomal subunit, (b) it is sensitive to edeine and aurintricarboxylic acid, and (c) the bound fMet-tRNAf is converted directly to fMet-puromycin upon addition of the large subunit. However, the eukaryotic reaction is GTPindependent.In this paper, we report that, in further analogy to IF-2 (8), the Artemia factor also promotes the poly(U)-dependent binding of N-acetylPhe-tRNA to 40S subunits, and the bound N-acylaminoacyl-tRNA is directly converted to N-acetylPhe-puromycin after addition of 60S subunits. Furthermore, the Artemia factor catalyzes, at low Mg2+ concentrations, not only the reported (1, 3, 5, 6) binding of Phe-tRNA, but also that of Met-tRNAf to 40S subunits.However, the unacylated derivatives are bound to a lesser extent than the N-acylated ones, and the bound aminoacyltRNA is less reactive with puromycin. The poly(U)-directed synthesis of N-acetylPhe-(Phe)n by the Artemia system requires (a) supernatant factor for formation of the 40S initiation complex, (b) the 60S subunit, elongation factor EF-1, and GTP, for binding of the second aminoacyl-tRNA and ensuing synthesis of the first peptide bond, and (c) elongation factor EF-2 for further growth of the chain...

Isolation and purification of hen oviduct protein synthesis initiation factors A2A and A2B

“…In all of these systems A2A and A2B were able to substitute for rabbit reticulocyte M2A and M2B. The molecular weights of Factors capable of initiating protein synthesis have been described in several eucaryotic species (Prichard et al, 1970;McCroskey et al, 1972;Picciano et al, 1973;Schrier and Staehelin, 1973; Levin et al, 1973;Grummt, 1974). Generally, these factors have been extracted from crude polysomes or microsomes with high concentrations of salt (0.5-1.0 M).…”

Conformational Control of the Interaction of Eukaryotic Elongation Factors EF-1 and EF-2 with Ribosomes

“…Evidence for the formation of stage 2 and 3 ribosomes was given previously (13 GTP to a specific radioactivity of 638 cpm/pmol. The source of other materials was as in previous work (8,9,13 (6) or E. coli (2)(3)(4)(5), the elongation factors EF-1 (EF-Tu) and EF-2 (EF-G) compete for a common ribosomal site or interact with closely overlapping sites, and (b) that such site(s) is (are) formed by association of the two ribosomal subunits.…”

“…For Ribosomal Complexes. These complexes were prepared on the basis of earlier work (13), with Artemia ribosomes or ribosomal subunits, using poly(U) as messenger, AcPhetRNA for chain initiation, and [14C]Phe-tRNA for chain elongation. The (7), leaves deacylated tRNAPhe on the P site.…”