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Pinpointing the interaction domains of plant seryl- tRNA synthetase and metabolic protein BEN1

Aminoacyl-tRNA synthetases (aaRS) support translational process by attaching appropriate amino acids to cognate tRNAs. Charged tRNA molecules reach ribosome and the amino acid can be transfered onto a growing peptide, according to the genetic code. It is well known that aaRSs can be involved in diverse cellular functions beyond translation independently or by forming protein- protein interactions. Understanding these non- canonical functions opens new perspectives in functional proteomics. However, the studies of plant aaRSs are scarce. Therefore our recent findings on interaction of BEN1, protein involved in metabolism of brassinosteroid hormones, and cytosolic seryl-tRNA synthetase (SerRS) from plant Arabidopsis thaliana broadens our knowledge of novel non-translational functions of plant aaRSs. We detected BEN1:SerRS interaction in vivo using Y2H screen and confirmed it in vitro using SPR method, also determining the dissociation constant of the protein complex. To pinpoint regions responsible for protein-protein interaction truncated variants of both SerRS and BEN1 protein were prepared and analyzed using microscale thermophoresis assay, that also enables Kd determination. Kd for complex containing BEN1 and truncated SerRS variant without basic C-terminal extension was similar to Kd of the SerRS:BEN1 complex indicating that basic C-terminal extension does not participate in interaction. Furthermore, N-terminal tRNA binding domain of SerRS did not form the complex with BEN1. Therefore we conclude that interaction involves the central part of SerRS that contains globular catalytic domain. Furthermore, using reverse transcription- quantitative polymerase chain reaction we investigated possible correlation of these two proteins on the level of gene expression in wild type plants, but also in transgenic plants overexpressing SerRS. We observed slightly lower expression of BEN1 gene in transgenic plants compared to wild type. Combining insights from both gene and protein level allowed us to define interaction surfaces and propose functional importance of SerRS:BEN1 assembly.

protein-protein interaction ; BEN1 ; plant seryl-tRNA synthetase ; Aminoacyl-tRNA synthetases ; Brassinosteroids

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