Properies of tRNAPhe from yeast carrying a spin label on the 3'-terminal. Interaction with yeast phenylalanyl-tRNA Synthetase and elongation factor Tu from Escherichia coli

Abstract

The 2-thioketo function of tRNAPhe-C-s2C-A in which the penultimate cytidine residue is replaced by 20thiocytidine can serve as a site of specific attachment of spin label. By alkylation of tRNAPhe-C-s2C-A with iodoacetamide or its spin label derivatives tRNAPhe-C-(acm)s2C-A or tRNAPheC-(SL)s2C-A are formed. The enzymatic phenylalanylation of these tRNAsPhe revealed that the 2-position of the penultimate cytidine can be modified without impairing this enzymatic reaction but there exists a sterical limitation for the subsituent on this position beyond which the tRNAPhe:phenylalanyl-tRNA synthetase recognition is not possible. Both Phe-tRNAPhe-C-(acm)s2C-A as well as Phe-tRNAPhe-C(SL)s2C-A form ternary complexes with EF-Tu.GTP. The part of the 3'-terminus of tRNAPhe where the additional substituents are attached is therefore not involved in the interaction with this elongation factor. This could be also demonstrated by ESR measurements of spin labelled tRNAsPhe. The correlation times, tauc, for tRNAPhe-C-(SL)s2C-A, Phe-tRNAPhe-C-(SL)s2C-A and Phe-tRNAPhe-C-(SL)s2C-A.EF-Tu:GTP are essentially identical indicating that the structure of the 3'-end of tRNAPhe is not influenced significantly by aminoacylation or ternary complex formation.