COMPETITION OF AMINOACYL-TRANSFER RNA-SYNTHETASES FOR TRANSFER-RNA ENSURES THE ACCURACY OF AMINOACYLATION

被引:47
作者
SHERMAN, JM [1 ]
ROGERS, MJ [1 ]
SOLL, D [1 ]
机构
[1] YALE UNIV, DEPT MOLEC BIOPHYS & BIOCHEM, NEW HAVEN, CT 06511 USA
关键词
D O I
10.1093/nar/20.11.2847
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The accuracy of protein biosynthesis rests on the high fidelity with which aminoacyl-tRNA synthetases discriminate between tRNAs. Correct aminoacylation depends not only on identity elements (nucleotides in certain positions) in tRNA (1), but also on competition between different synthetases for a given tRNA (2). Here we describe in vivo and in vitro experiments which demonstrate how variations in the levels of synthetases and tRNA affect the accuracy of aminoacylation. We show in vivo that concurrent overexpression of Escherichia coli tyrosyl-tRNA synthetase abolishes misacylation of supF tRNA(Tyr) with glutamine in vivo by overproduced glutaminyl-tRNA synthetase. In an in vitro competition assay, we have confirmed that the overproduction mischarging phenomenon observed in vivo is due to competition between the synthetases at the level of aminoacylation. Likewise, we have been able to examine the role competition plays in the identity of a non-suppressor tRNA of ambiguous identity, tRNA(Glu). Finally, with this assay, we show that the identity of a tRNA and the accuracy with which it is recognized depend on the relative affinities of the synthetases for the tRNA. The in vitro competition assay represents a general method of obtaining qualitative information on tRNA identity in a competitive environment (usually only found in vivo) during a defined step in protein biosynthesis, aminoacylation. In addition, we show that the discriminator base (position 73) and the first base of the anticodon are important for recognition by E. coli tyrosyl-tRNA synthetase.
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页码:2847 / 2852
页数:6
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