Detection of Noncovalent tRNA·Aminoacyl-tRNA Synthetase Complexes by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Ita Gruic-Sovulj,Hans-Christian Lüdemann,Franz Hillenkamp,Ivana Weygand-Durasevic,Zeljko Kucan,Jasna Peter-Katalinic

doi:10.1074/jbc.272.51.32084

Abstract

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS) was used for the study of complexes formed by yeast seryl-tRNA synthetase (SerRS) and tyrosyl-tRNA synthetase (TyrRS) with tRNASer and tRNATyr. Cognate and noncognate complexes were easily distinguished due to a large mass difference between the two tRNAs. Both homodimeric synthetases gave MS spectra indicating intact desorption of dimers. The spectra of synthetase-cognate tRNA mixtures showed peaks of free components and peaks assigned to complexes. Noncognate complexes were also detected. In competition experiments, where both tRNA species were mixed with each enzyme only cognate alpha2.tRNA complexes were observed. Only cognate alpha2.tRNA2 complexes were detected with each enzyme. These results demonstrate that MALDI-MS can be used successfully for accurate mass and, thus, stoichiometry determination of specific high molecular weight noncovalent protein-nucleic acid complexes.

Highlights

Matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-MS) was used for the study of complexes formed by yeast seryl-tRNA synthetase (SerRS) and tyrosyl-tRNA synthetase (TyrRS) with tRNASer and tRNATyr
These results demonstrate that MALDI-MS can be used successfully for accurate mass and, stoichiometry determination of specific high molecular weight noncovalent protein-nucleic acid complexes
Our present study extends the application of mass spectrometry, in general, and MALDI-MS, in particular, to studies of biologically relevant protein-nucleic acid complexes with

Summary

Introduction

Matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-MS) was used for the study of complexes formed by yeast seryl-tRNA synthetase (SerRS) and tyrosyl-tRNA synthetase (TyrRS) with tRNASer and tRNATyr. Cognate ␣21⁄7tRNA2 complexes were detected with each enzyme These results demonstrate that MALDI-MS can be used successfully for accurate mass and, stoichiometry determination of specific high molecular weight noncovalent protein-nucleic acid complexes. As indicated by crystal structures of several synthetase1⁄7tRNA pairs, members of each synthetase class interact with two different regions of the L-shaped tRNA molecules [7] The formation of both cognate and non-cognate synthetase1⁄7tRNA complexes has been observed by a variety of experimental techniques such as. The two yeast aminoacyl-tRNA synthetases are representatives of class I and class II synthetases, respectively Their amino acid sequences based on gene structures are known [18, 19], and crystal structures of their bacterial counterparts have been determined at high resolution [5, 20, 21]. The specific noncovalent complex of transcription factor, PU. DNA-binding domain with a 17-base pairs double-stranded DNA fragment, has been analyzed, providing an example for the study of a biologically relevant protein-nucleic acid complex [31]

Methods

Results

Conclusion