Equivalent and Non‐equivalent Binding Sites for tRNA on Aminoacyl‐tRNA Synthetases

Abstract

Complexes between tRNAPhe (yeast), tRNAser (yeast) and tRNATy' (Escherichia coli) and their cognate aminoacyl-tRNA synthetases have been studied by sedimentation velocity runs in an analytical ultracentrifuge. The amount of complex formation was determined by the absorption and the sedimentation coefficients of the fast-moving boundary in the presence of excess tRNA or excess synthetase respectively. The same method has been applied to unspecific combinations of tRNAs and synthetases. Inactive material of tRNA or synthetase does not influence the results.1. Two moles of tRNAPhe can be bound to one mole of phenylalanyl-tRNA synthetase with a binding constant % lo6 M-I. The binding constants for both tRNAs are very similar; the binding sites are independent of each other. Omission of Mg2+ does not prevent binding.2. Two moles of tRNASer can be bound to one mole of Seryl-tRNA synthetase; the binding of the first and second tRNA is non-equivalent. K , % lo6 M-' , K2 is determined to be 1.3 x lo5 M-' at pH 7.2. Omission of Mg2 + prevents complex formation. 3. Tyrosyl-tRNA synthetase behaves very similarly to seryl-tRNA synthetase. The binding constant for the weakly bound tRNA is 2 . 3~ lo5 M-' at pH 7.2, and 2 . 5~ lo6 M-' at pH 6.0. No complexes are observed in the absence of Mg2+.4. Unspecific binding was only obtained with phenylalanyl-tRNA synthetase. It binds tRNASFr (yeast), tRNAA'" (yeast) and tRNATyr (E. coli) with a binding constant about 100 times lower compared to its cognate tRNA.The binding data are discussed with respect to the tertiary structure of the tRNAs, the subunit structure of the synthetases and the possible physical basis for the non-equivalence of binding sites.Aminoacyl-tRNA synthetases functionally represent a unique class of enzymes. They differ, however, substantially in their physical properties and the mechanistic details of the reaction they catalyze. It has been of particular interest to correlate the number of binding sites for the various substrates as well as the number of catalytic sites to the size of the synthetases and their subunit structures. So far, molecular weights ranging from 50000 to 250000 have been determined. Subunit structures of the sll, cx2, aB and a2P2 type have been found (review: [l]). Up till now no generalization could be made as to a correspondence of size, number of identical subunits, and number of binding or catalytic sites.In the present paper we will concern ourselves merely with the binding of tRNA to the aminoacyltRNA synthetases. In most cases studied so far only one binding site was found per synthetase. This includes large enzymes like the phenylalanyl-tRNA synthetase from Escherichia coli [2,3], which has an sl,~,-structure, as well as small synthetases like the glutaminyl-tRNA synthetase from E. coli [4], which has an a,-structure. (1975)

Anticooperative Binding of l‐Tryptophan to Tryptophanyl‐tRNA Synthetase from Beef Pancreas

“…This is in contrast with the data that had been previously obtained in different experimental conditions of temperature and pH [lo]. It has been shown that changes in pH [15], temperature [16] or proteolysis [17] may alter the binding properties of synthetases. Therefore it was made sure that a non-proteolyzed form of tryptophanyl-tRNA synthetase was used throughout this study.…”

Structural Asymmetry of the Terminal Catalytic Complex in Selenocysteine Synthesis

“…A number of oligomeric aaRS have previously been shown to exhibit activity of half of the sites despite containing multiple, seemingly chemically equivalent active and tRNA-binding sites (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)30). The mode of tRNA binding described herein argues that SepSecS may follow the sequential model of allosteric regulation (49).…”

“…These enzymes exhibit "half of the sites" activity that is presumably regulated by negative allostery. The TyrRS dimer binds one tRNA Tyr (15) and catalyzes formation of 1 mol of tyrosyl adenylate (16 -20), and tetrameric PheRS (15,21) and SepRS (22) employ two of their four catalytic and tRNA-binding sites at a time. Likewise, class II enzymes, bacterial LysRS-II (23,24), AspRS (25), and HisRS (26) and the archaeal and eukaryotic ProRS (27,28) behave according to the "half of the sites" model.…”

Binding of tRNA to Escherichia coli Ribosomes as Measured by Velocity Sedimentation

“…The tRNA boundaries were compared in the presence and absence of ribosomes. A similar method has been used extensively with other interacting macromolecular systems such as tRNA and tRNA ligases; it has been described in detail elsewhere [20]. The method is even more simple in the system used in the present work because the sedimentation coefficients of free and bound tRNA are different by more than an order of magnitude (except with 30-S subunits).…”