Mammalian polypeptide chain release factor and tryptophanyl-tRNA synthetase are distinct proteins.

Abstract

A very high (approximately 90%) structural similarity exists between the bovine, human and murine tryptophanyl‐tRNA synthetases (WRS), and quite unexpectedly the rabbit polypeptide chain release factor (eRF). This similarity may point to a very close resemblance or identity between these proteins involved in distinct steps of protein synthesis, or inadvertently to an incorrect assignment of the clone reported to encode eRF, since the structure of clones encoding WRS were confirmed by peptide sequencing. Using high resolution column chromatography and sucrose gradient centrifugation combined with assays for WRS and eRF activities, we show that functionally distinct WRS and eRF proteins can be completely separated from each other. Moreover, a putative anti‐eRF monoclonal antibody appears incapable of immunoprecipitating the eRF activity or binding to protein(s) possessing eRF activity. This antibody binds to protein fractions which coincide in various separation procedures with rabbit WRS activity, and to pure bovine WRS. The protein expressed in Escherichia coli from the original cDNA clone initially reported to encode eRF, has WRS activity but not eRF activity. Resequencing of the fragment of the original rabbit cDNA demonstrates the presence of the previously overlooked HXGH motif typical of class I aminoacyl‐tRNA synthetases. Consequently, mammalian WRS and eRF are different proteins, and the cDNA clone formerly assigned as encoding eRF encodes rabbit WRS.

Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3.

“…If termination of translation in prokaryotes and eukaryotes is similar, one may anticipate the existence of a second eRF, since no GTP binding motifs were identified in eRFI (Frolova et al, 1994), while it has been demonstrated earlier that in mammals termination of translation is a GTP-dependent process (Beaudet and Caskey, 1971;Konecki et al, 1977). Furthermore, we have observed, after the last step of rabbit eRFI purification (Frolova et al, 1993b(Frolova et al, , 1994, that its activity decreases significantly and becomes GTP independent.…”

From past to future: suppressor mutations in yeast genes encoding translation termination factors

“…The cloning of cDNA encoding the RF of a rabbit was reported in 1990 (Lee et al, 1990). A surprisingly high degree of similarity between the protein and tryptophanyl-tRNA synthetase identified served as evidence that the identified gene encodes the rabbit tryptophanyl synthetase but not the RF factor (Frolova et al, 1993). Even in early experiments, translation termination in mammals was demonstrated to be a GTP-dependent process (Beaudet and Caskey, 1971;Konecki et al, 1977), but to isolate a protein with GTPase activity was not possible.…”

Polypeptide chain release factors

“…Although the presence of a rabbit reticulocyte release factor (eRF) was first shown 25 years ago, only recently have the protein and the gene been correctly identified. A gene previously thought to code for eRF (Lee et al, 1990) was finally shown to encode rabbit tryptophanyl-tRNA synthetase (Frolova et al, 1993). At present, it appears that a single polypeptide in eukaryotes (eRF1) fulfils the role of both of the prokaryotic factors RF1 and RF2, i.e.…”