Interaction of different tRNAs with translation elongation factors 1A from lower and higher eukaryotes

P V Futernyk,B S Negrutskii,A V El'Skaya

doi:10.7124/bc.0007f8

Abstract

Aim. The work is aimed at confirmation of earlier assumed mechanism of tRNA channeling. Methods. The methods of band shift assay and Forsters resonance energy transfer were used. Results. The affinities of mammalian tRNAs for two tissue specific isoforms of elongator factor eEF1A1 and eEF1A2 were compared. For the first time we have shown the ability of yeast eEF1A*GDP to form non-canonical ternary complex with deacylated tRNAs. The complexation of eukaryotic eEF1A with initiator tRNA, of both bacterial and mammalian origin, was also demonstrated. Conclusions. The formation of the non-canonical complexes of eEF1A*GDP with deacylated tRNAs is common for higher and lower eukaryotes, which is in favor of universality of eukaryotic tRNA-channeling.

Highlights

The process of protein biosynthesis is known to be functionally compartmentalized [1], and it is known that tRNA is never free in the cell and all the time is bound to some partners [2, 3]
We have examined the ability of a number of various deacylated tRNAs to form ternary complex with two tissue specific isoforms of eEF1A by band shift assay [4,5,6]
We used the same technique for the comparative analysis of the ability of bacterial, yeast and mammalian elongation factors 1A to bind deacylated [32P]-labeled tRNAs. tRNAVal, tRNATyr, tRNALys, tRNAiMet from rabbit liver, tRNAifMet from E. coli and tRNALeu from T. thermophilus were used

Summary

Introduction

The process of protein biosynthesis is known to be functionally compartmentalized [1], and it is known that tRNA is never free in the cell and all the time is bound to some partners [2, 3]. The protein eEF1A forms a complex with aa-tRNA in a GTP-dependent manner and delivers it to the ribosomal A-site. In higher eukaryotes eEF1A*GDP is suggested to accept deacylated tRNA from the ribosomal E-site delivering it to aaRSes for recharge [1]. It was found that the affinity of tissue specific isoforms of the factor, eEF1A1 and eEF1A2, to tRNA is different. KD for the complexes of eEF1A2 with tRNAs usually is 2–4 fold lower than that for eEF1A1 [6]. Of interest is a relatively high affinity of eEF1A to deacylated tRNAs (KD ≤750 nM) in comparison with prokaryotic EF1A (KD > 2200 nM [7,8,9]). A role of the tRNA body in the ternary complex formation was discussed for years [10,11,12]

Methods

Results

Conclusion