Jenvert Rose-Marie, Schiavone Lovisa Holmberg
School of Life Sciences, Södertörns högskola, S-141 04 Huddinge, Sweden.
J Mol Biol. 2007 Jan 19;365(3):764-72. doi: 10.1016/j.jmb.2006.10.065. Epub 2006 Oct 25.
The stringent response is activated by the binding of stringent factor to stalled ribosomes that have an unacylated tRNA in the ribosomal aminoacyl-site. Ribosomes lacking ribosomal protein L11 are deficient in stimulating stringent factor. L11 consists of a dynamic N-terminal domain (amino acid residues 1-72) connected to an RNA-binding C-terminal domain (amino acid residues 76-142) by a flexible linker (amino acid residues 73-75). In vivo data show that mutation of proline 22 in the N-terminal domain is important for initiation of the stringent response. Here, six different L11 point and deletion-mutants have been constructed to determine which regions of L11 are necessary for the activation of stringent factor. The different mutants were reconstituted with programmed 70 S(DeltaL11) ribosomes and tested for their ability to stimulate stringent factor in a sensitive in vitro pppGpp synthesis assay. It was found that a single-site mutation at proline 74 in the linker region between the two domains did not affect the stimulatory activity of the reconstituted ribosomes, whereas the single-site mutation at proline 22 reduced the activity of SF to 33% compared to ribosomes reconstituted with wild-type L11. Removal of the entire linker between the N and C-terminal domains or removal of the entire proline-rich helix beginning at proline 22 in L11 resulted in an L11 protein, which was unable to stimulate stringent factor in the ribosome-dependent assay. Surprisingly, the N-terminal domain of L11 on its own activated stringent factor in a ribosome-dependent manner without restoring the L11 footprint in 23 S rRNA in the 50 S subunit. This suggests that the N-terminal domain can activate stringent factor in trans. It is also shown that this activation is dependent on unacylated tRNA.
严谨反应是由严谨因子与核糖体A位点上带有未氨酰化tRNA的停滞核糖体结合所激活的。缺乏核糖体蛋白L11的核糖体在刺激严谨因子方面存在缺陷。L11由一个动态的N端结构域（氨基酸残基1 - 72）通过一个柔性接头（氨基酸残基73 - 75）连接到一个RNA结合C端结构域（氨基酸残基76 - 142）组成。体内数据表明，N端结构域中脯氨酸22的突变对于严谨反应的启动很重要。在此，构建了六种不同的L11点突变体和缺失突变体，以确定L11的哪些区域对于严谨因子的激活是必需的。不同的突变体与编程的70S（ΔL11）核糖体进行重组，并在灵敏的体外pppGpp合成测定中测试它们刺激严谨因子的能力。结果发现，两个结构域之间接头区域中脯氨酸74的单点突变不影响重组核糖体的刺激活性，而脯氨酸22的单点突变使严谨因子的活性相较于用野生型L11重组的核糖体降低至33%。去除N端和C端结构域之间的整个接头或去除L11中从脯氨酸22开始的整个富含脯氨酸的螺旋，会产生一种L11蛋白，该蛋白在核糖体依赖性测定中无法刺激严谨因子。令人惊讶的是，L11的N端结构域自身以核糖体依赖性方式激活严谨因子，而没有恢复50S亚基中23S rRNA的L11足迹。这表明N端结构域可以反式激活严谨因子。还表明这种激活依赖于未氨酰化的tRNA。
