Era and RbfA have overlapping function in ribosome biogenesis in Escherichia coli
Era and RbfA have overlapping function in ribosome biogenesis in Escherichia coli
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DOI：
10.1159/000092818
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发表时间：
2006-01-01
影响因子：
1.2
通讯作者：
Inouye, Masayori
中科院分区：
文献类型：
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作者：
Inoue, Koichi;Chen, Jingqiu;Inouye, Masayori
一种冷休克蛋白RbfA(核糖体结合因子A)是细胞在低温下生长所必需的。在RbfA缺失株中，30S和50S核糖体亚基相对于70S单体增加，伴随着前体16S rRNA(17S RRNA)的积累。最近，我们报道了一种必需的GTP结合蛋白Era的过表达不仅抑制了RbfA缺失菌株的冷敏感细胞生长，而且还抑制了核糖体的生物合成。在这里，为了阐明RbfA和era如何在功能上重叠，我们鉴定了一个冷敏感Era突变体(Glu-200到Lys的点突变；E200K)，它表现出与RbfA缺失菌株相似的表型：游离核糖体亚基和17S rRNA的积累。为了检测E200K在RbfA缺失菌株中的作用，我们构建了E200K诱导表达系统。有趣的是，与野生型era不同的是，在RbfA缺失菌株中过表达era(E200K)蛋白严重抑制了细胞的生长，即使在允许的温度下也会伴随16S rRNA的进一步减少。纯化的E1a(E200K)蛋白与30S核糖体亚基结合的方式与野生型Era一样依赖于核苷酸，并保留GTP酶和自磷酸化活性。此外，我们还分离到了E200K突变体的自发返回体。这些反转体部分抑制了17S rRNA的积累。所有自发突变均可导致E200K基因的高表达。这些结果表明，Era(E200K)蛋白在核糖体生物发生中功能受损，但不失去其核糖体结合活性。在RbfA缺失株中，E200K引起的严重生长缺陷可能是由于内在野生型E200K与过度表达的E200K竞争结合30S核糖体亚基所致。我们认为Era和RbfA具有重叠的功能，这对核糖体的生物发生是必不可少的，并且RbfA只有在高温下才变得可有可无，因为Era只有在更高的温度下才能补充其功能。版权所有(C)2006 S.Karger AG，巴塞尔。
A cold-shock protein, RbfA (ribosome-binding factor A), is essential for cell growth at low temperature. In an rbfA-deletion strain, 30S and 50S ribosomal subunits increase relative to 70S monosomes with concomitant accumulation of a precursor 16S rRNA (17S rRNA). Recently, we have reported that overexpression of Era, an essential GTP-binding protein, suppresses not only the cold-sensitive cell growth but also defective ribosome biogenesis in the rbfA-deletion strain. Here, in order to elucidate how RbfA and Era functionally overlap, we characterized a cold-sensitive Era mutant (a point mutation at the Glu-200 to Lys; E200K) which shows a similar phenotype as the rbfA-deletion strain; accumulation of free ribosome subunits and 17S rRNA. To examine the effect of E200K in the rbfA-deletion strain, we constructed an E200K-inducible expression system. Interestingly, unlike wildtype Era, overexpression of Era(E200K) protein in the rbfA-deletion strain severely inhibited cell growth even at permissive temperature with further concomitant reduction of 16S rRNA. Purified Era(E200K) protein binds to 30S ribosomal subunits in a nucleotide-dependent manner like wild-type Era and retains both GTPase and autophosphorylation activities. Furthermore, we isolated spontaneous revertants of the E200K mutant. These revertants partially suppressed the accumulation of 17S rRNA. All the spontaneous mutations were found to result in higher Era(E200K) expression. These results suggest that the Era(E200K) protein has an impaired function in ribosome biogenesis without losing its ribosome binding activity. The severe growth defect caused by E200K in the rbfA-deletion strain may be due to competition between intrinsic wild-type Era and over expressed Era(E200K) for binding to 30S ribosomal subunits. We propose that Era and RbfA have an overlapping function that is essential for ribosome biogenesis, and that RbfA becomes dispensable only at high temperatures because Era can complement its function only at higher temperatures. Copyright (c) 2006 S. Karger AG, Basel.