Research paperAn archaeal aminoacyl-tRNA synthetase complex for improved substrate quality control
Introduction
Aminoacyl-tRNA synthetases (aaRSs) catalyze the first step in protein biosynthesis, the esterification of their cognate tRNAs with appropriate amino acids [1]. To increase the processivity of translation, some aaRSs form complexes with other components of the translation machinery, i.e. other aaRSs, elongation factors and the ribosome [[2], [3], [4], [5]]. Additionally, to expand beyond this, canonical role in protein synthesis, aaRSs frequently associate with other cellular macromolecules that may not be involved in translation [3,5].
In methanogenic archaeon Methanothermobacter thermautotrophicus arginyl- (ArgRS) and seryl-tRNA synthetase (SerRS) establish a transient non-covalent complex [6,7]. In eukaryotic organisms both SerRS and ArgRS interact with other cellular proteins to form complexes of varying stability [8]. SerRS is associated with the translasome, a supercomplex composed of aaRSs, ribosomal proteins, elongation factors and the proteasome in fission yeast [9] and transcription factor Yin Yang 1 in human cells [10]. ArgRS has been shown to form complexes with other tRNA synthetases in Caenorhabditis elegans [11] and mammalia [4].
A multisynthetase complex (MSC) of M. thermautotrophicus is composed of leucyl-, lysyl- and prolyl-tRNA synthetases and its interaction with the elongation factor EF-1α [12,13] indicates a potential coupling between tRNA aminoacylation and aminoacyl-tRNA decoding [12]. Likewise, both SerRS and ArgRS make a direct interaction with the large ribosomal subunit in M. thermautotrophicus [6]. Both ArgRS and SerRS associate with the ribosomal proteins of the L7/L12 stalk and proteins near the stalk base of the 50S subunit. The proximity of the ribosomal A-site may suggest that these interactions improve the efficiency of Arg-tRNAArg and Ser-tRNASer transfer, from respective synthetases to EF-1α and, subsequently, to the ribosome. Also, overrepresented consecutive usage of synonymous codons for serine and arginine indicates that ribosomal co-localization of the SerRS•ArgRS complex involves purposeful recharging of tRNA isodecoders within the polysomes [6].
Binary ArgRS•SerRS interaction promotes serylation reaction [7] and enhances SerRS•tRNASer complex formation [14]. ArgRS was shown to increase SerRS's affinity for cognate tRNA two-fold, but did not affect cooperative properties or the complex’ stoichiometry [14]. Driven by these findings, here we set up to determine the topological determinants of the SerRS•ArgRS interaction and its functional relevance in tRNA arginylation. We produced several ArgRS variants bearing sequential deletions of the structural, N-terminal elements (Fig. 1A) and inspected their capacity to participate in SerRS•ArgRS complex formation by various approaches. Furthermore, we assessed the participation of the cognate substrates in order to reveal existence of stable ternary complexes. Finally, we produced several posttranscriptional variants of tRNAArg and showed the influence that SerRS exerts on arginylation of these substrates.
Section snippets
Protein expression and purification
Full length SerRS-His6 and GST-ArgRS were expressed and purified essentially as described [7]. For production of the tagless SerRS, pET28 vector containing serS gene [7] was digested with NcoI and NdeI, blunted using Klenow polymerase and P1 nuclease and ligated. To generate N-terminally His-tagged wild type ArgRS, argS sequence was inserted between BamHI and XhoI sites of a pET28b vector. Truncated ArgRS variants were generated by PCR using pET28b-argS as template and T7 promoter/terminator
Determinants that govern ArgRS participation in SerRS•ArgRS assembly
To identify the structural elements of ArgRS that participate in ArgRS•SerRS complex assembly, we generated truncated ArgRS variants and measured their affinity for full length SerRS. Truncations were introduced according to our structural model (Fig. 1A, S1) and their stability was assessed by fluorescence and circular dichroism measurements (Fig. 1A, S2, S3).
The largest truncations eliminated entire N- or C-domains (variants ΔNtot and ΔCtot, Fig. 1A, S3). Removal of the N-terminal domain
Discussion
Here we show that ArgRS uses its N-terminal domain to establish interactions with both SerRS and cognate tRNAArg in a highly analogous manner. In both cases helix H4 (missing in ΔNtot-ArgRS, but not in ΔN89-ArgRS) exerts the largest influence on the complex formation (Table 1). Although this element seems to establish direct contacts with SerRS, it does not participate directly in tRNA recognition, as evidenced by our structural model of MtArgRS bound to tRNAArg [31]. Instead, helix H4 acts as
Conclusion
Using pull down and SPR analysis, we show that (i) ArgRS uses its N-terminal domain to establish contacts with both SerRS and cognate tRNAArg and that (ii) only SerRS′ substrates participate in the formation of stable ternary complexes (i.e. [SerRS•Ser-AMP]•ArgRS and [SerRS•tRNASer]•ArgRS). ArgRS does not distinguish between posttranscriptional variants of tRNAArg and catalyzes arginylation of these substrates with the same efficiency. Aminoacylation of the naïve, unmodified tRNAArg can be
Contributions
AC, GA and IWĐ conceived and designed the experiments. AC, MČ and VGM performed the experiments. AC, IWĐ and IGS wrote the manuscript, whereas all authors read, edited and approved the final manuscript.
Acknowledgements
We thank Nediljko Budiša for access to the research facilities at the Technische Universität Berlin. We are grateful to Vesna Hodnik for her advice on SPR experiments. This work is supported by the grants from Croatian Science Foundation (Grant 09.01/293), ARRS BI-HR/12-13-025 Molecular interactions in protein synthesis and European Community's Seventh Framework Programme (FP7RegPot/IntegraLife, 315997).
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Prof. Weygand-Đurašević is deceased.