Protein Initiation in Eukaryotes: Formation and Function of a Ternary Complex Composed of a Partially Purified Ribosomal Factor, Methionyl Transfer RNA _f , and Guanosine Triphosphate

Abstract

A protein factor contained in a 1 M KCI extract of L-cell ribosomes and partially purified by chromatography on DEAE-cellulose forms a specific ternary complex with rat-liver Met-tRNAf and GTP. The complex is measured by its quantitative retention on nitrocellulose membranes. Complex assembly is optimal at 100 mM KC1 and 0.2 mM MgCl2, and is independent of mRNA and of ribosomes. The GTP requirement can be replaced over 65% by its methylene analogue GDPCH2P, indicating that GTP hydrolysis is not involved. Complex formation is inhibited by 10 MM aurintricarboxylic acid, but is unaffected by 100 MM pactamycin, 100 MM fusidic acid, or by excess uncharged methionine tRNAf. The ternary complex is relatively stable and appears at the void volume during filtration on Sephadex G-100. At 1-3 mM MgCl2 and in the presence of other factors, the ternary complex is implicated in protein initiation by (i) its capacity to bind to the 40S ribosomal subunit to form a 48S complex; and (ii) the subsequent association of the 48S complex with a 60S subunit to form a functional "80S complex."There is considerable evidence to suggest that in eukaryotes, the rate-determining step in protein synthesis occurs during the initiation phase of translation. Such a rate-limiting step has been observed in such diverse tissues as cells in mitosis (1), muscles of diabetic animals (2), livers of fasted animals (3, 4), as well as in cell cultures exposed to aminoacid-deficient medium (5, 6), temperature changes (7), and alterations in sodium-potassium ratios (8). Other studies in both prokaryotes and eukaryotes indicate that the sequence of events leading to the formation of an 80S initiation complex is mediated by the stepwise participation of specific ribosome factors (9)(10)(11)(12)(13)(14). Consequently, the first steps in the initiation sequence are of particular importance if it is assumed that they represent logical sites for the operation of regulatory controls.Recent studies inI prokaryotic systems have rel)orted that one of the earliest steps in the initiation sequence is the formation of a ternary complex containing the ribosome initiation factor and GTP (15,16).In this report, we describe the in vitro formation of a eukaryotic ternary complex with similar properties, composed of a partially purified ribosome factor from L-929 mouse fibroblasts (L-cells), rat-liver Met-tRNAf, and GTP. Preliminary data were presented in a previous communication (17

Polypeptide chain initiation in eukaryotes: initiation factor MP in Artemia salina embryos.

“…Our results with A. salina IF-MP are in full accord with those reported with factor from other sources (13)(14)(15)(16)(17)(18)(19)(20) (29).…”

“…As seen in Table 1, IF-MP activity as measured by ternary complex formation with A. salina [35S]Met-tRNAj, is very low in undeveloped embryos but increases markedly during development. The factor is present in both the highspeed supernatant and the ribosomal wash fraction to the extent of about 30% and 70% of the total, respectively, but its specific activity is about 50-fold higher in the ribosomal wash. Properties of factor As found for IF-MP from other sources (13)(14)(15)(16)(17)(18)(19)(20)27), the retention of eukaryotic Met-tRNAr by nitrocellulose filters in the presence of the A. salina factor is markedly GTP-dependent (Fig. 1).…”

Eukaryotic and archaeal translation initiation factor 2: A heterotrimeric tRNA carrier

“…Eukaryotic/archaeal initiation factor 2 (e/aIF2) has been characterized 30 years ago as a protein which forms a ternary complex with GTP and methionylated initiator tRNA, and subsequently binds to the small ribosomal subunit (e.g. [1–7]). eIF2 is composed of three subunits called α, β and γ [5,7], coded by SUI2 [8,9], SUI3 [10] and GCD11 [11] genes, respectively.…”

Preparation and activity of synthetic unmodified mammalian tRNAi Met in initiation of translation in vitro

“…Translation of eukaryotic mRNA is initiated by a unique aminoacyl-tRNA, Met-tRNA i Met (Housman et al+, 1970;Smith & Marcker, 1970), which passes through a complex series of highly specific interactions with components of the translation apparatus before the methionine residue attached to this initiator tRNA is incorporated into a protein+ Eukaryotic initiator tRNAs are 76 nt long and contain eight residues that have additional base modifications (Fig+ 1B)+ The tRNA i Met moiety is aminoacylated by methionyl tRNA synthetase (MetRS) and Met-tRNA i Met is then specifically bound by eukaryotic initiation factor 2 (eIF2) to form a stable ternary complex with GTP (Dettman & Stanley, 1972;Levin et al+, 1973) that subsequently binds to the 40S ribosomal subunit with eIF3 in an mRNA-independent reaction, resulting in formation of a 43S preinitiation complex (Darnborough et al+, 1973;Schreier & Staehelin, 1973)+ The 43S complex binds mRNA in either a 59-capdependent manner or by direct association with an internal ribosomal entry site (IRES) within the 59-untranslated region (59 UTR; Jackson, 2000)+ Initiator tRNA occupies the ribosomal P-site in the 48S complex and is directly involved in mediating selection of the start site of translation by the ribosome as it scans in a 59-39 direction on the 59 UTR (Cigan et al+, 1988;Pestova & Hellen, 1999)+ After base pairing between the initiation codon and the anticodon of initiator tRNA has been established, eIF5 and eIF5B mediate displacement of initiation factors from the 40S subunit and joining of a 60S ribosomal subunit to the residual [40S/MettRNA i Met ] complex (Pestova et al+, 2000a(Pestova et al+, , 2000b)+ The aminoacyl terminus of initiator tRNA is placed in the peptidyltransferase center of the resulting 80S ribosome, a second (elongator) tRNA is placed in the ribosomal A-site by eukaryotic elongation factor 1A (eEF1A) and the first peptide bond is formed+ AUG (methionine) codons at internal positions within an open reading frame are not recognized by tRNA i…”