γ‐Glutamyl Phosphate Attached to Glutamine‐Specific tRNA

Abstract

B. subtilis Gln‐tRNA formation, in vitro , involves the initial acceptance of glutamic acid by tRNA Gln to form a missense Glu‐tRNA Gln intermediate which is converted to Gln‐tRNA by a subsequent amidation step, catalyzed by a specific amido‐transferase, and requiring divalent cations, ATP, and l ‐glutamine or l ‐asparagine as amide donor. This reaction is associated with stoichiometric cleavage of glutamine and ATP to yield glutamic acid and P i , respectively. Amidation proceeds via the formation of an activated intermediate which is shown to be γ‐phospho‐Glu‐tRNA Gln ( P ‐γ‐Glu‐tRNA Gln ). P ‐γ‐Glu‐tRNA Gln , bound to amido‐transferase, is detected following incubation in the absence of amide donor. Subsequent addition of l ‐glutamine to the system leads to the rapid loss of the phosphate moiety from the intermediate concomitantly with the formation of Gln‐tRNA, which is released from the enzyme. Consequences of the pathway, if it is duplicated in vivo , are the separation of the synthesis of glutamine destined for protein from that of free glutamine and, further, the coupling of the synthesis of the amino acid with that of protein. These implications are discussed with regard to their bearing on possible functions of the pathway.