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

大肠杆菌RNLA是核糖核酸酶LS的必需基因。事实上，His标记的RNLA在体外显示出内分泌停滞活性。然而，与体内相比，这种活性过于简单和低。在细胞提取液中发现的RNase LS活性的鉴定表明，该RNase由一个大的复合体组成，其质量估计为1000 kDa。事实上，西方印迹强烈暗示希斯-鲁拉参与了这一复杂的活动。我们用镍珠拉下His-RNLA寻找与His-RNLA相关的蛋白质，并通过氨基酸测序确定了包括TpiA和SucB在内的12个RNLA相关蛋白质。因为核糖核酸酶LS活性因tpiA或suB的中断而受损。我们的结论是，1000 kDa的复合体是RNaseLS的组成部分。RNLB位于大肠杆菌基因组图谱中RNLA的下游。当我们干扰RNLB时，突变体没有表现出RNase LS活性。此外，从细胞提取液中下拉His-RNLb也是…更多的RNLA被镍小球沉淀，这些蛋白质是用咪唑从小球中洗脱出来的。这些结果有力地表明RNLB也参与了1000 kDa的复合体。RNase LS以前被认为依赖于mRNA的翻译能力来切割mRNA。在这项研究中，我们发现RNase LS根据翻译终止过程切割终止密码子下游的mRNA，我们发现在T4感染后立即激活RNase E和G来迅速降解宿主mRNAs。这种降解需要T4基因的表达，因为在T4感染之前加入利福平的情况下，这种快速降解不会发生。为了寻找致病基因T4，我们对T4的几个缺失突变体进行了检测，发现致病基因与TK2区有关。由于TK2区含有20个未知基因，我们将TK2区分为4个亚区，并制备了缺失每个亚区或亚区组合的缺失突变体。利用缺失突变体，我们发现RNaseE的激活需要其中一个亚区，RNaseG的激活需要四个亚区中的两个。因此，核糖核酸酶E和G由一组不同的T4基因激活。较少

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