Shigeno Yuta, Uchiumi Toshio, Nomura Takaomi
Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567, Japan.
Department of Biology, Faculty of Science, Niigata University, Niigata 950-2181, Japan.
Biochem Biophys Res Commun. 2016 Apr 22;473(1):237-242. doi: 10.1016/j.bbrc.2016.03.085. Epub 2016 Mar 19.
Ribosomal protein L6, an essential component of the large (50S) subunit, primarily binds to helix 97 of 23S rRNA and locates near the sarcin/ricin loop of helix 95 that directly interacts with GTPase translation factors. Although L6 is believed to play important roles in factor-dependent ribosomal function, crucial biochemical evidence for this hypothesis has not been obtained. We constructed and characterized an Escherichia coli mutant bearing a chromosomal L6 gene (rplF) disruption and carrying a plasmid with an arabinose-inducible L6 gene. Although this ΔL6 mutant grew more slowly than its wild-type parent, it proliferated in the presence of arabinose. Interestingly, cell growth in the absence of arabinose was biphasic. Early growth lasted only a few generations (LI-phase) and was followed by a suspension of growth for several hours (S-phase). This suspension was followed by a second growth phase (LII-phase). Cells harvested at both LI- and S-phases contained ribosomes with reduced factor-dependent GTPase activity and accumulated 50S subunit precursors (45S particles). The 45S particles completely lacked L6. Complete 50S subunits containing L6 were observed in all growth phases regardless of the L6-depleted condition, implying that the ΔL6 mutant escaped death because of a leaky expression of L6 from the complementing plasmid. We conclude that L6 is essential for the assembly of functional 50S subunits at the late stage. We thus established conditions for the isolation of L6-depleted 50S subunits, which are essential to study the role of L6 in translation.
核糖体蛋白L6是大亚基（50S）的重要组成部分，主要与23S rRNA的螺旋97结合，并位于螺旋95的肌动蛋白/蓖麻毒素环附近，该环直接与GTPase翻译因子相互作用。尽管人们认为L6在因子依赖性核糖体功能中起重要作用，但尚未获得支持这一假设的关键生化证据。我们构建并鉴定了一个大肠杆菌突变体，该突变体的染色体L6基因（rplF）被破坏，并携带一个带有阿拉伯糖诱导型L6基因的质粒。尽管这个ΔL6突变体的生长速度比其野生型亲本慢，但它在阿拉伯糖存在的情况下仍能增殖。有趣的是，在没有阿拉伯糖的情况下细胞生长呈双相性。早期生长仅持续几代（LI期），随后是几个小时的生长停滞（S期）。这个停滞期之后是第二个生长阶段（LII期）。在LI期和S期收获的细胞含有因子依赖性GTPase活性降低的核糖体，并积累了50S亚基前体（45S颗粒）。45S颗粒完全缺乏L6。无论L6缺失情况如何，在所有生长阶段都观察到了含有L6的完整50S亚基，这意味着ΔL6突变体因互补质粒中L6的渗漏表达而逃过死亡。我们得出结论，L6对于后期功能性50S亚基的组装至关重要。因此，我们建立了分离L6缺失的50S亚基的条件，这对于研究L6在翻译中的作用至关重要。
