Analysis of the function of E-coli 23S rRNA helix-loop 69 by mutagenesis -: art. no. 18
Analysis of the function of E-coli 23S rRNA helix-loop 69 by mutagenesis -: art. no. 18
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DOI：
10.1186/1471-2199-6-18
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发表时间：
2005-07-29
影响因子：
--
通讯作者：
Remme, J
中科院分区：
文献类型：
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作者：
Liiv, A;Karitkina, D;Remme, J
背景：核糖体是一种双亚基酶，在蛋白质合成过程中表现出结构动力学。亚基间桥已被提出在解码、易位和肽基转移酶反应中发挥重要作用，但其贡献的物理性质尚不清楚。一个有趣的亚基间桥，B2a，其中包含23S rRNA螺旋69作为主要成分，已牵连的接近在一些催化重要的区域。除了接触小核糖体亚基，螺旋69同时接触A和P位点的tRNA和几个translation factors.Results：我们扫描的螺旋69环的诱变和分析的突变体核糖体使用质粒携带IPTG诱导的表达系统。我们分析了23S rRNA突变对细胞生长的影响，突变体核糖体对细胞多核糖体库的贡献以及突变体核糖体在无细胞翻译中发挥作用的能力。突变A1912 G和A1919 G具有非常强的生长表型，在体外蛋白质合成过程中无活性，并且在多核糖体中表达不足。突变Psi 1917 C具有非常强的生长表型，并导致细胞多核糖体库的普遍耗尽。结论：23SrRNA的A1912G、A1919G和Psi1917C突变对70S核糖体的组装有明显的抑制作用。突变A1916G导致50S亚基或70S形成缺陷。突变Psi 1911 C、A1913 G、C1914 A、Psi 1915 C和A1918 G缺乏明确的表型。
Background: The ribosome is a two-subunit enzyme known to exhibit structural dynamism during protein synthesis. The intersubunit bridges have been proposed to play important roles in decoding, translocation, and the peptidyl transferase reaction; yet the physical nature of their contributions is ill understood. An intriguing intersubunit bridge, B2a, which contains 23S rRNA helix 69 as a major component, has been implicated by proximity in a number of catalytically important regions. In addition to contacting the small ribosomal subunit, helix 69 contacts both the A and P site tRNAs and several translation factors.Results: We scanned the loop of helix 69 by mutagenesis and analyzed the mutant ribosomes using a plasmid-borne IPTG-inducible expression system. We assayed the effects of 23S rRNA mutations on cell growth, contribution of mutant ribosomes to cellular polysome pools and the ability of mutant ribosomes to function in cell-free translation. Mutations A1912G, and A1919G have very strong growth phenotypes, are inactive during in vitro protein synthesis, and under-represented in the polysomes. Mutation Psi 1917C has a very strong growth phenotype and leads to a general depletion of the cellular polysome pool. Mutation A1916G, having a modest growth phenotype, is apparently defective in the assembly of the 70S ribosome.Conclusion: Mutations A1912G, A1919G, and Psi 1917C of 23S rRNA strongly inhibit translation. Mutation A1916G causes a defect in the 50S subunit or 70S formation. Mutations Psi 1911C, A1913G, C1914A, Psi 1915C, and A1918G lack clear phenotypes.