THE ROLE OF SPERMINE IN PREVENTING MISACYLATION BY PHENYLALANYL-TRANSFER RNA-SYNTHETASE

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  • 1 January 1981
    • journal article
    • research article
    • Vol. 256  (13) , 6729-6735
Abstract
Physiological concentrations of polyamines contribute significantly to both the speed and precision of aminoacylation of tRNA. Unphysiologically high concentrations of Mg are required to obtain high rates of synthesis of .**GRAPHIC**. catalyzed by yeast phenylalamine:tRNA ligase in vitro. Under such conditions, rates of misacylation, e.g., the synthesis of .**GRAPHIC**. may be 1/5 of the rate of correct acylation. High rates of correct aminoacylation are achieved in the presence of physiological concentrations of Mg (1.0 mM) plus spermine (0.2 mM). Under these conditions, there is almost no misacylation. A kinetic study of the Mg dependence of aminoacylation shows that the rate-determining transition state for Phe-tRNAPhe synthesis contains, in addition to tightly bound Mg2+, either 2 spermines (Kd = .apprx. 50 .mu.M) or 2 Mg2+ ions (Kd = .apprx. 1.0 mM). The tRNAPhe binds 2 spermines to form a very compact, very precisely defined structure that easily forms an activated E.cntdot.S [enzyme.cntdot.substrate] conformer, E.cntdot.S* (Jencks'' Circe Effect). In the absence of spermine, 2 Mg2+ ions bind more poorly to the tRNA to form a similar but not identical tRNA conformer which, is less able to form E.cntdot.S* and more slowly aminoacylated. Misacylation of noncognate tRNA requires several additional, more loosely bound Mg2+ ions that serve to relax the tRNA structure. Such Mg driven relaxation has a minor effect on Km (tRNA) but the resulting floppy structure is able to avoid the barriers against reaction and is aminoacylated thousands of times more rapidly than the compact defined structure.