University of Tartu, Institute of Technology, Nooruse 1, 50411 Tartu, Estonia.
Department of Molecular Biology, Umeå University, Building 6K, 6L, SE-901 87 Umeå, Sweden.
Nucleic Acids Res. 2018 Feb 28;46(4):1973-1983. doi: 10.1093/nar/gky023.
During amino acid starvation the Escherichia coli stringent response factor RelA recognizes deacylated tRNA in the ribosomal A-site. This interaction activates RelA-mediated synthesis of alarmone nucleotides pppGpp and ppGpp, collectively referred to as (p)ppGpp. These two alarmones are synthesized by addition of a pyrophosphate moiety to the 3' position of the abundant cellular nucleotide GTP and less abundant nucleotide GDP, respectively. Using untagged native RelA we show that allosteric activation of RelA by pppGpp increases the efficiency of GDP conversion to achieve the maximum rate of (p)ppGpp production. Using a panel of ribosomal RNA mutants, we show that the A-site finger structural element of 23S rRNA helix 38 is crucial for RelA binding to the ribosome and consequent activation, and deletion of the element severely compromises (p)ppGpp accumulation in E. coli upon amino acid starvation. Through binding assays and enzymology, we show that E. coli RelA does not form a stable complex with, and is not activated by, deacylated tRNA off the ribosome. This indicates that in the cell, RelA first binds the empty A-site and then recruits tRNA rather than first binding tRNA and then binding the ribosome.
在氨基酸饥饿的情况下，大肠杆菌严谨反应因子 RelA 识别核糖体 A 位中的去酰化 tRNA。这种相互作用激活了 RelA 介导的警报核苷酸 pppGpp 和 ppGpp 的合成，统称为（p）ppGpp。这两种警报核苷酸是通过在丰富的细胞核苷酸 GTP 和较不丰富的核苷酸 GDP 的 3' 位置添加焦磷酸部分合成的。使用未标记的天然 RelA，我们表明 pppGpp 对 RelA 的别构激活增加了 GDP 转化为实现（p）ppGpp 产生的最大速率的效率。使用一组核糖体 RNA 突变体，我们表明 23S rRNA 螺旋 38 的 A 位指状结构元件对于 RelA 与核糖体的结合和随后的激活至关重要，并且该元件的缺失严重损害了氨基酸饥饿时大肠杆菌中（p）ppGpp 的积累。通过结合测定和酶学，我们表明大肠杆菌 RelA 不会与核糖体上的去酰化 tRNA 形成稳定的复合物，也不会被其激活。这表明在细胞中，RelA 首先结合空的 A 位，然后招募 tRNA，而不是先结合 tRNA，然后结合核糖体。
