Purification and properties of isoleucyl ribonucleic acid synthetase from Escherichia coli.

Aminoacyl ribonucleic acid formation is the first step in the translation of the genetic code for protein synthesis (1, 2). Although base pairing between the aminoacyl-RNA and the ribosome-bound messenger RNA may explain the mechanism of “reading” the messenger RNA base sequence, very little is known about how the transfer RNA structure is “read” by the activating enzymes. This, of course, is quite a different problem, for it involves specific interactions between a protein and a polynucleotide. As yet, there is no other example of enzymes that must distinguish between a large number of similar yet distinct nucleic acid molecules. To learn more about this type of specific interaction, we need to know at least as much about the structure of the enzyme as we now know about the structure of transfer RNA. To date, a number of aminoacyl-RNA synthetases have been partially purified (3-13), but relatively little attention has been given to their physical properties and the parameters determining their specific biological activity. With this objective in mind, we have purified the isoleucyl-RNA synthetase from Escherichia coli (14). In addition, this enzyme has the interesting property of being able to form valyladenylate as well as isoleucyladenylate, although only the enzyme-bound isoleucyl-AMP serves as an intermediate in the synthesis of aminoacyl-RNA. In this paper, we describe the detailed purification of the enzyme and certain of its properties. The accompanying paper (15) is concerned with the mechanism of amino acid activation, the interaction of the RNA with the enzyme aminoacyladenylate complex, and in particular, the fate of the enzii, . . . Val-AMP1 complex in the presence of tRNAii,.