In mammalian cells valyl-tRNA synthetase (ValRS) forms a high M r complex with the four subunits of elongation factor EF-1H. The ␤, ␥, and ␦ subunits, that contribute the guanine nucleotide exchange activity of EF-1H, are tightly associated with the NH 2 -terminal polypeptide extension of valyl-tRNA synthetase. In this study, we have examined the possibility that the functioning of the companion enzyme EF-1␣ could regulate valyl-tRNA synthetase activity. We show here that the addition of EF-1␣

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... nd GTP in excess in the aminoacylation mixture is accompanied by a 2-fold stimulation of valyl-tRNA Val synthesis catalyzed by the valyl-tRNA synthetase component of the ValRS⅐EF-1H complex. This effect is not observed in the presence of EF-1␣ and GDP or EF-Tu⅐GTP and requires association of valyl-tRNA synthetase within the ValRS⅐EF-1H complex. Since valyl-tRNA synthetase and elongation factor EF-1␣ catalyze two consecutive steps of the in vivo tRNA cycle, aminoacylation and formation of the ternary complex EF-1␣⅐GTP⅐Val-tRNA Val that serves as a vector of tRNA from the synthetase to the ribosome, the data suggest a coordinate regulation of these two successive reactions. The EF-1␣⅐GTP-dependent stimulation of valyl-tRNA synthetase activity provides further evidence for tRNA channeling during protein synthesis in mammalian cells. 1 The abbreviations used are: ValRS, valyl-tRNA synthetase; DTE, 1,4-dithioerythritol; GMP-PNP, ␤;␥-imidoguanosine 5Ј-triphosphate.