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Aminoacyl-tRNA synthetases and ribosomes: tracing the roots of interactions by in vivo and quantitative biophysical methodS for interactome analysis

It has been estimated that over 80% of proteins do not operate alone but in complexes. Examples of important complexes are the spliceosome, the ribosome and the nuclear pore complex. The ribosome is a ribonucleoprotein complex that translates the genetic information in the cell into the encoded polypeptides. The fidelity with which mRNA is decoded into proteins is essential for maintenance of the genetic code and it is highly dependent on the specific attachment of amino acids to tRNAs by aminoacyl-tRNAsynthetases (aaRSs). Multi- aminoacyl-tRNAsynthetase complexes of different compositions occur throughout the tree of life. To further investigate the extent and composition of multi-aaRS complexes in archaea we have undertaken a yeast two-hybrid search for proteins that interact with methanogenic- type SerRS (mSerRS), an atypical form of SerRS confined to certain archaea such as Methanothermobacter thermautotrophicus (M. thermautotrophicus). Yeast two-hybrid screens revealed a interaction between two aaRSs: mSerRS and ArgRS, which appears to specifically enhance tRNASer aminoacylation. Moreover, in the same organism, few ribosomal proteins were found also associated with mSerRS. Chemical crosslinking and surface plasmon resonance (SPR) experiments confirmed that mSerRS and ArgRS associate with large ribosomal subunit proteins (1). Microscale thermophoresis (MST) was used to determine dissociation constant (Kd) for the fluorescently labeled ribosomal particles in interaction with mSerRS and MtArgRS consistent with formation of a stable macromolecular complex detected by ultracentrifugation. A general feature of the ribosome is the large subunit stalk protuberance which consists of 4–6 copies (2–3 dimers) of ribosomal protein P1 (L12) where the elongation factors bind and act. We consider ribosomal protein P1 to be a primary target for interaction of mSerRS with the ribosome and thus we exploited the intrinsic trypthophan SerRS fluorescence to study the interaction with wild type and mutant variants of L12 by fluorescence spectroscopy. Obtained dissociation constants for mSerRS:L12 complexes were comparable to Kds determined by MST analysis. In addition, the assembly of mSerRS with M. thermautotrophicus ribosome is supported by analysis of the ordering of synonymous codons. In M. thermautotrophicus found that the codon choice at the next instance of the same amino acid may be influenced by the previous codon upstream, causing autocorrelation of codon pairs read by the same tRNA. Therefore, the association of components of the protein synthesis machinery into macromolecular assemblies has the potential to increase translational efficiency by limiting substrate diffusion away from the ribosome thereby allowing rapid recycling of tRNAs. [1] Godinic-Mikulcic, Vlatka ; Jaric, Jelena ; Greber, Basil ; Franke, Vedran ; Hodnik, Vesna ; Anderluh, Gregor ; Ban, Nenad and Weygand- Durasevic, Ivana (2014) Archaeal aminoacyl-tRNA synthetases interact with the ribosome to recycle tRNAs. Nucl. Acids Res. 42 (8):5191- 5201.

protein interactions; fluorescence spectrophotometry; microscale thermophoresis; dissociation constant; protein synthesis; ribosome; two-hybrid system; surface plasmon resonance

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