engleski

The aminoacyl-tRNA synethetase complex in archaea

Aminoacyl-tRNA synthetases (aaRS) mediate the conversion of genetic information stored in nucleotide sequence of a mRNA molecule to an amino acid sequence of a growing polypeptide chain by synthetizing aminoacyl-tRNAs, building blocks for protein biosynthesis. In all three domains of life, these enzymes show a tendency to organize in higher order complexes either with other proteins involved in the process of translation or with cellular components that operate within processes beyond translation. Complexes of this kind can vary in size and number of diverse functions they are involved in, ranging from large (1×106 Da) multisynthetase complexes (MSCs) characteristic for higher eukaryotes harbouring 9 aminoacylation activities to simple bacterial binary complexes that contain one aaRS and some other protein factor, coupled to perform tasks such as tRNA editing or metabolite synthesis. Archaeal MSCs exhibit intermediate complexity and usually encompass more than one aminoacylation activity. In archaeon Methanothermobacter thermautotrophicus, several MSCs have been identified, using different aaRSs as baits in several yeast two-hybrid screens. Seryl-tRNA synthetase:arginyl-tRNA synthetase (SerRS:ArgRS) interaction determined in one of these screens has been shown to enhance aminoacylation of cognate tRNA by SerRS 2- to 3, 5-fold. In attempt to crystallize such binary complex, we decided to proceed with the same pair of aaRSs from another methanogenic archaeon, Methanosarcina barkeri, given the high sequence homology between corresponding synthetases, as well as better overall yields in protein purification procedure. Interactions were confirmed in vitro by a pull- down assay, as well as chemical crosslinking implying a 2:1 ArgRS:SerRS stoichiometry. Kinetic analysis of the interaction investigated by means of surface plasmon resonance yielded a 300 nM KD suggesting high affinity between aforementioned aaRSs. However, the interaction is also characterized by very high rates of association and dissociation, hampering the possibility of stable complex isolation using gel filtration. Seven commercial and several grid screens have been used in attempt of finding optimal crystallization conditions in constellation with different molar ratios of aaRSs. Crystals were observed in more than 50 different conditions but unfortunately contained single aaRS. Nature of the interaction may impose necessity to modulate kinetic parameters employing genetic engineering in order to obtain the structure of SerRS:ArgRS complex. Since none of the MSCs has been crystallized yet, structure od this complex would provide a valuable information.

aminoacyl-tRNA synthetases; multisynthetase complex; crystallization

nije evidentirano