PylSn and the Homologous N-terminal Domain of Pyrrolysyl-tRNA Synthetase Bind the tRNA That Is Essential for the Genetic Encoding of Pyrrolysine |
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Authors: | Ruisheng Jiang Joseph A. Krzycki |
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Affiliation: | From the ‡Ohio State Biochemistry Program.;§Center for RNA Biology, and ;the ¶Department of Microbiology, The Ohio State University, Columbus, Ohio 43210 |
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Abstract: | Pyrrolysine is represented by an amber codon in genes encoding proteins such as the methylamine methyltransferases present in some Archaea and Bacteria. Pyrrolysyl-tRNA synthetase (PylRS) attaches pyrrolysine to the amber-suppressing tRNAPyl. Archaeal PylRS, encoded by pylS, has a catalytic C-terminal domain but an N-terminal region of unknown function and structure. In Bacteria, homologs of the N- and C-terminal regions of archaeal PylRS are respectively encoded by pylSn and pylSc. We show here that wild type PylS from Methanosarcina barkeri and PylSn from Desulfitobacterium hafniense bind tRNAPyl in EMSA with apparent Kd values of 0.12 and 0.13 μm, respectively. Truncation of the N-terminal region of PylS eliminated detectable tRNAPyl binding as measured by EMSA, but not catalytic activity. A chimeric protein with PylSn fused to the N terminus of truncated PylS regained EMSA-detectable tRNAPyl binding. PylSn did not bind other D. hafniense tRNAs, nor did the competition by the Escherichia coli tRNA pool interfere with tRNAPyl binding. Further indicating the specificity of PylSn interaction with tRNAPyl, substitutions of conserved residues in tRNAPyl in the variable loop, D stem, and T stem and loop had significant impact in binding, whereas those having base changes in the acceptor stem or anticodon stem and loop still retained the ability to complex with PylSn. PylSn and the N terminus of PylS comprise the protein superfamily TIGR03129. The members of this family are not similar to any known RNA-binding protein, but our results suggest their common function involves specific binding of tRNAPyl. |
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Keywords: | Amino Acid Aminoacyl tRNA Synthetase Archaea Bacteria RNA-binding Protein Transfer RNA (tRNA) 22nd Amino Acid Genetic Code Pyrrolysine Pyrrolysyl-tRNA Synthetase |
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