伪尿苷(Φ),尿苷的异构体,通常在所有生物体的非编码RNA的不同位置发现。Φ残基是由许多单或多位点特异性Φ合成酶形成的,它们通常充当独立的蛋白质。此外,在尤卡里亚和古细菌,特异性核糖核蛋白复合物,每个都包含一个不同的盒H/ACA指导RNA和四个核心蛋白,可以在不同的细胞RNA的许多位点产生Φ。Cbf5是这些复合物中的核心Φ合酶。使用Haloferax火山作为古细菌模型生物,我们证明,与真核生物相反,Cbf5同源物(HVO_2493)在该古细菌中不是必需的。H.vancolii的Cbf5缺失菌株在其23SrRNA的位置1940、1942、2605和2591(大肠杆菌位置1915、1917、2572和2586)处完全缺乏Φ,并且含有降低的稳态水平的一些盒H/ACARNA。已知古细菌Cbf5在体外具有tRNAVL55合酶活性,但我们无法在H.volculii体内证实这种活性。相反,Pus10(以前是PsuX)同源物(HVO_1979),它可以在体外产生tRNAVL55,以及VL54,在这里被证明是在H.火山中必不可少的,而相应的tRNAVL55合酶,Pus4和TruB,在酵母和大肠杆菌中不是必需的,分别。最后,我们证明了HVO_1852,TruA/Pus3同源物,负责H.volcaniitRNA中39位的假尿苷化,并且相应的基因不是必需的。
Pseudouridine (Ψ), the isomer of uridine, is commonly found at various positions of noncoding RNAs of all organisms. Ψ residues are formed by a number of single- or multisite specific Ψ synthases, which generally act as stand-alone proteins. In addition, in Eukarya and Archaea, specific ribonucleoprotein complexes, each containing a distinct box H/ACA guide RNA and four core proteins, can produce Ψ at many sites of different cellular RNAs. Cbf5 is the core Ψ synthase in these complexes. Using Haloferax volcanii as an archaeal model organism, we show that, contrary to eukaryotes, the Cbf5 homolog (HVO_2493) is not essential in this archaeon. The Cbf5-deleted strain of H. volcanii completely lacks Ψ at positions 1940, 1942, 2605, and 2591 (Escherichia coli positions 1915, 1917, 2572, and 2586) of its 23S rRNA, and contains reduced steady-state levels of some box H/ACA RNAs. Archaeal Cbf5 is known to have tRNA Ψ55 synthase activity in vitro but we could not confirm this activity in vivo in H. volcanii. Conversely, the Pus10 (previously PsuX) homolog (HVO_1979), which can produce tRNA Ψ55, as well as Ψ54 in vitro, is shown here to be essential in H. volcanii, whereas the corresponding tRNA Ψ55 synthases, Pus4 and TruB, are not essential in yeast and E. coli, respectively. Finally, we demonstrate that HVO_1852, the TruA/Pus3 homolog, is responsible for the pseudouridylation of position 39 in H. volcanii tRNAs and that the corresponding gene is not essential.