Artificial regulation of gene expression in Escherichia coli by RNase P.

Plasmids encoding various external guide sequences (EGSs) were constructed and inserted into Escherichia coli. In strains harboring the appropriate plasmids, the expression of fully induced beta-galactosidase and alkaline phosphatase activity was reduced by more than 50%, while no reduction in such activity was observed in strains with non-specific EGSs. The inhibition of gene expression was virtually abolished at restrictive temperatures in strains that were temperature-sensitive for RNase P (EC 3.1.26.5). Northern blot analysis showed that the steady-state copy number of EGS RNAs was several hundred per cell in vivo. A plasmid that contained a gene for M1 RNA covalently linked to a specific EGS reduced the level of expression of a suppressor tRNA that was encoded by a separate plasmid. Similar methods can be used to regulate gene expression in E. coli and to mimic the properties of cold-sensitive mutants. IMAGES:

构建了编码多种外源向导序列（external guide sequences, EGSs）的质粒，并将其转化至大肠杆菌（Escherichia coli）中。在携带对应质粒的菌株中，经完全诱导后的β-半乳糖苷酶与碱性磷酸酶活性均下降超过50%；而携带非特异性EGSs的菌株则未观察到此类酶活性的降低。对于核糖核酸酶P（RNase P，EC 3.1.26.5）温度敏感型菌株，其基因表达抑制作用在限制性培养温度下几乎完全被消除。Northern印迹杂交分析显示，体内EGS RNA的稳态拷贝数可达每细胞数百个。一种携带与特定EGSs共价连接的M1 RNA基因的质粒，可降低另一质粒所编码的抑制型转运RNA（suppressor tRNA）的表达水平。类似方法可用于调控大肠杆菌的基因表达，并模拟冷敏感突变株的特性。IMAGES: