Equivalent and non-equivalent binding sites for tRNA on aminoacyl-tRNA synthetases.
Complexes between tRNAPhe (yeast), tRNASer (yeast) and tRNATyr (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 greater than 10(6) M-1. 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, K1 greater than 10(6) M-1, K2 is determined to be 1.3 X 10(5) M-1 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 X 10(5) M-1 at pH 7.2, and 2.5 X 10(6) M-1 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 tRNASer (yeast), tRNAAla (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.
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008433:Mathematics; D008970:Molecular Weight; D010649:Phenylalanine; D010652:Phenylalanine-tRNA Ligase / Q000378:metabolism; D011485:Protein Binding; D012343:RNA, Transfer / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012694:Serine; D012714:Serine-tRNA Ligase / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine; D014448:Tyrosine-tRNA Ligase / Q000378:metabolism
D046911:Macromolecular Substances; D014443:Tyrosine; D012694:Serine; D010649:Phenylalanine; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D014448:Tyrosine-tRNA Ligase; D012714:Serine-tRNA Ligase; D010652:Phenylalanine-tRNA Ligase; D008274:Magnesium