Effects of the ligands of beef tryptophanyl-tRNA synthetase on the elementary steps of the tRNATrp aminoacylation

Abstract

Tryptophanyl-tRNA synthetase catalyzed formation of Trp-tRNA(Trp) has been studied by mixing tRNA(Trp) with a preformed bis(tryptophanyl adenylate)-enzyme complex in the 0-60-ms time range, on a quenched-flow apparatus. Analyzing the data gives an association rate constant ka = (1.22 +/- 0.47) X 10(8) M-1 S-1, a dissociation rate constant kd = 143 +/- 73 S-1, and a dissociation constant Kd = 1.34 +/- 0.80 microM for tRNA(Trp). The maximum rate constant of tryptophan transfer to tRNA(Trp) is kt = 33 +/- 3 S-1. When starting the aminoacylation reaction with a mono(tryptophanyl adenylate)-enzyme complex, one obtains different kinetic profiles than when using a bis(tryptophanyl adenylate)-enzyme complex. Over a 0-400-ms time range, the monoadenylate-enzyme complex yields an apparent first-order reaction, while the bis-adenylate-enzyme complex yields a biphasic aminoacylation of tRNA(Trp). Analysis of Trp-tRNA(Trp) formation from both complexes according to simple reaction schemes shows that the dissociation of tRNA(Trp) from an enzyme subunit carrying no adenylate is 6.9-fold slower than from an enzyme subunit carrying an adenylate. The apparent rate constant of dissociation of nascent tryptophanyl-tRNA(Trp) is 4.9 S-1 in the absence of free tryptophan, which is much slower than its rate of formation (33 S-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Novel complexes of mammalian translation elongation factor eEF1A·GDP with uncharged tRNA and aminoacyl‐tRNA synthetase

“…Taking into account rather low affinity of tRNA for the E site of 80S ribosomes (the apparent K d is about 600 n m [33]), it is plausible to assume that the transfer of tRNA from the E site to eEF1A·GDP occurs due to the affinity gradient ( K d for [eEF1A·GDP·tRNA] is 20 n m , this study). Furthermore, the ARS affinity for [eEF1A·GDP·tRNA] ( K d is 9 n m , this study) is higher than that for free tRNA ( K d in the range of 100–200 n m [34,35]), which makes association of the enzyme with tRNA bound to eEF1A·GDP thermodynamically favorable. In this quaternary complex, a transfer of tRNA from the factor to ARS may occur.…”

Two conformations of a crystalline human tRNA synthetase–tRNA complex: implications for protein synthesis

“…Tryptophan was shown to bind competitively with charged tRNA Trp (Trp‐tRNA Trp ) to bovine TrpRS (Trezeguet et al , 1986). As a consequence, Trp promoted dissociation of nascent Trp‐tRNA Trp from bovine TrpRS (Merle et al , 1988). (Similarly, in the related class I IleRS, at least three independent experiments showed that, after aminoacylation, the binding of a new molecule of isoleucine promoted the dissociation of Ile‐tRNA Ile (Yarus and Berg, 1969; Eldred and Schimmel, 1972; Eldred and Schimmel, 1973). )…”

Application of capillary zone electrophoresis to the study of interactions betweenBacillus subtilis tryptophanyl-tRNA synthetase and tRNATrp

“…The calculated number of sites was 1.14; the dissociation constant was calculated to be 0.63 pmol L -1 (95% confidence intervals (C.I. ): 0,51-0.75 pmol L-l), which is of the same order of magnitude as the value of 0.19 pmol L -1 for interactions of beef TrpRS and tRNATrp [12,15].…”

“…We assume that tRNATrp transfers between TrpRS and TrpRS-tRNATrp complex quickly, and no stable complex exists. Merle also thought it likely that the association process of beef TrpRS and tRNATrp is comparatively fast [12]. Figure 4 shows schemes of the interactions.…”