A new turn in the research on RNA metabolism of Escherichia coli

大肠杆菌rnlA是RNase LS的必需基因。事实上，His标记的RnlA在体外表现出内核糖核酸酶活性。然而，这种活性过于简单，与体内相比，活性较低。在细胞提取物中发现的RNA酶LS活性的表征显示，该RNA酶由大的复合物组成，其质量估计为1000 kDa。事实上，蛋白质印迹法强烈表明His-RulA参与了该复合体。我们用镍珠拉下His-RnlA以找到与His-RnlA相关的蛋白，并通过氨基酸测序确定了包括TpiA和SucB在内的12个RnlA相关蛋白。因为RNase LS活性被tpiA或sucB的破坏所损害。我们得出结论，1000-kDa复合物是RNaseLS的组成部分。rnlB在E. coli基因组图谱。当我们破坏rnlB时，突变体没有表现出RNase LS活性。此外，从细胞提取物中拉下His-RnlB也 ...更多信息 通过Ni珠沉淀RnlA，并且用咪唑从珠上共洗脱这些蛋白质。这些结果有力地表明，RnlB也参与了1000-kDa复合物。在本研究中，我们确定了RNase LS依赖于翻译终止过程切割终止密码子下游的mRNA。我们发现，在T4感染后，RNase E和G立即被激活，迅速降解宿主mRNA。这种降解需要T4基因的表达，因为在T4感染前加入利福平的情况下不会发生这种快速降解。为了寻找致病的T4基因，我们测试了几个T4的缺失突变体，发现致病基因涉及TK 2区域。由于TK 2区域包含20个未知基因，我们将该区域分为4个亚区，并制备缺失每个亚区或亚区组合的缺失突变体。使用缺失突变体，我们发现RNase E的激活需要一个子区域，RNase G的激活需要四个子区域中的两个。因此，RNA酶E和G被不同的T4基因组激活。少

Escherichia coil rnlA was identified as an essential gene for RNase LS. In fact, His-tagged RnlA exhibited an endoribonulcease activity in vitro. However, this activity was too simple and low in comparison with that in vivo. Characterization of RNase LS activity found in a cell extract revealed that this RNase consisted of a large complex, the mass of which was estimated 1000 kDa. In fact, His-RulA was strongly suggested by western blotting to be involved in the complex. We pulled down His-RnlA with Ni-beads to find proteins associated with His-RnlA and twelve RnlA-associating proteins including TpiA and SucB were determined by aminoacid sequencing. Because the RNase LS activity was impaired by disruption of tpiA or sucB. we conclude that the 1000-kDa complex is an integral part of RNase LS. rnlB locates immediately downstream of rnlA in the E. coli genomic map. When we disrupted rnlB, the mutant exhibited no RNase LS activity. In addition, pull-down of His-RnlB from cell extract also … More precipitated RnlA by Ni-beads, and these proteins were coeluated from the beads with imidazole. These results strongly suggests that RnlB is also involved in the 1000-kDa complex.RNase LS was formerly suggested to cleave mRNA depending translational ability of the mRNA. In this research, we established that RNase LS cleaves mRNA downstream of a termination codon depending on a translation termination process.We found that immediately after T4 infection, RNases E and G are activated to degrade host mRNAs rapidly. This degradation required the expression of T4 genes, because such rapid degradation did not occurr in the presence of rifampicin added in prior to T4 infection. In order to search for the causal T4 genes, we tested several deletion mutants of T4 and found that the causal genes are involved in TK2 region. Since TK2 region contains 20 unknown genes, we divided this region into 4 sub-regions and prepared deletion mutants that lacked each sub-region or a combination of sub-regions. Using the deletin mutants, we found that activation of RNase E required one of the sub-regions and that activation of RNase G required two of four sub-regions. Thus, RNases E and G are activated by a different set of T4 genes. Less