Roovers Martine, Hale Caryn, Tricot Catherine, Terns Michael P, Terns Rebecca M, Grosjean Henri, Droogmans Louis
Institut de Recherches Microbiologiques J.-M. Wiame, Avenue E. Gryson, 1 B-1070 Bruxelles, Belgium.
Nucleic Acids Res. 2006;34(15):4293-301. doi: 10.1093/nar/gkl530. Epub 2006 Aug 18.
Pseudouridine (Psi) located at position 55 in tRNA is a nearly universally conserved RNA modification found in all three domains of life. This modification is catalyzed by TruB in bacteria and by Pus4 in eukaryotes, but so far the Psi55 synthase has not been identified in archaea. In this work, we report the ability of two distinct pseudouridine synthases from the hyperthermophilic archaeon Pyrococcus furiosus to specifically modify U55 in tRNA in vitro. These enzymes are (pfu)Cbf5, a protein known to play a role in RNA-guided modification of rRNA, and (pfu)PsuX, a previously uncharacterized enzyme that is not a member of the TruB/Pus4/Cbf5 family of pseudouridine synthases. (pfu)PsuX is hereafter renamed (pfu)Pus10. Both enzymes specifically modify tRNA U55 in vitro but exhibit differences in substrate recognition. In addition, we find that in a heterologous in vivo system, (pfu)Pus10 efficiently complements an Escherichia coli strain deficient in the bacterial Psi55 synthase TruB. These results indicate that it is probable that (pfu)Cbf5 or (pfu)Pus10 (or both) is responsible for the introduction of pseudouridine at U55 in tRNAs in archaea. While we cannot unequivocally assign the function from our results, both possibilities represent unexpected functions of these proteins as discussed herein.
位于转运RNA（tRNA）第55位的假尿苷（Ψ）是在生命的所有三个域中都几乎普遍保守的RNA修饰。这种修饰在细菌中由TruB催化，在真核生物中由Pus4催化，但到目前为止，古菌中的Ψ55合成酶尚未被鉴定出来。在这项工作中，我们报告了来自嗜热古菌激烈火球菌的两种不同的假尿苷合成酶在体外特异性修饰tRNA中U55的能力。这些酶是（pfu）Cbf5，一种已知在rRNA的RNA引导修饰中起作用的蛋白质，以及（pfu）PsuX，一种以前未被表征的酶，它不是假尿苷合成酶的TruB/Pus4/Cbf5家族的成员。（pfu）PsuX此后重新命名为（pfu）Pus10。这两种酶在体外都特异性修饰tRNA U55，但在底物识别上表现出差异。此外，我们发现在异源体内系统中，（pfu）Pus10有效地补充了缺乏细菌Ψ55合成酶TruB的大肠杆菌菌株。这些结果表明，（pfu）Cbf5或（pfu）Pus10（或两者）很可能负责古菌tRNA中U55处假尿苷的引入。虽然我们不能从我们的结果中明确确定其功能，但这两种可能性都代表了本文所讨论的这些蛋白质的意外功能。
