PylSn and the Homologous N-terminal Domain of Pyrrolysyl-tRNA Synthetase Bind the tRNA That Is Essential for the Genetic Encoding of Pyrrolysine
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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DOI：
10.1074/jbc.m112.396754
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发表时间：
2012-09-21
影响因子：
4.8
通讯作者：
Krzycki, Joseph A.
中科院分区：
文献类型：
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作者：
Jiang, Ruisheng;Krzycki, Joseph A.
吡咯碱由编码蛋白质的基因中的琥珀色密码子表示，例如某些古细菌和细菌中存在的甲基胺甲基转移酶。吡咯基-TRNA合成酶（PYLRS）将吡咯氨氨酸连接到琥珀色抑制tRNA（PYL）。由PYLS编码的古细菌具有催化C末端结构域，但是功能和结构未知的N末端区域。在细菌中，古细菌的N-和C末端区域的同源物分别由PYLSN和PYLSC编码。我们在这里表明，来自甲状腺菌Barkeri的野生型PYL和pylsn的hafniense hafniense结合tRNA（PYL）分别为0.12和0.13 mu m，在EMSA中。塔的N末端区域的截断，消除了通过EMSA测量的可检测的tRNA（PYL）结合，而不是催化活性。与拟合的Pylsn的aChimeric蛋白融合到截短的塔的N末端，恢复了EMSA可检测的tRNA（PYL）结合。 Pylsn没有结合其他D. hafniense trNA，也没有结合大肠杆菌tRNA池的竞争会干扰tRNA（pyl）结合。进一步表明PyLSN与tRNA（pyl）的特异性，在变量环，d茎和t茎和环中的tRNA（pyl）中保守残基的取代对结合具有显着影响或反密码子茎和环仍然保留了与Pylsn复合的能力。 PYLSN和塔的N末端包括蛋白质超家族TIGR03129。该家族的成员与任何已知的RNA结合蛋白都不相似，但是我们的结果表明它们的共同功能涉及tRNA的特定结合（PYL）。
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 tRNA(Pyl). 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 tRNA(Pyl) in EMSA with apparent K-d values of 0.12 and 0.13 mu M, respectively. Truncation of the N-terminal region of PylS eliminated detectable tRNA(Pyl) binding as measured by EMSA, but not catalytic activity. Achimeric protein with PylSn fused to the N terminus of truncated PylS regained EMSA-detectable tRNA(Pyl) binding. PylSn did not bind other D. hafniense tRNAs, nor did the competition by the Escherichia coli tRNA pool interfere with tRNA(Pyl) binding. Further indicating the specificity of PylSn interaction with tRNA(Pyl), substitutions of conserved residues in tRNA(Pyl) 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 tRNA(Pyl).