Varshney U, RajBhandary U L
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
J Bacteriol. 1992 Dec;174(23):7819-26. doi: 10.1128/jb.174.23.7819-7826.1992.
We showed recently that a mutant of Escherichia coli initiator tRNA with a CAU-->CUA anticodon sequence change can initiate protein synthesis from UAG by using formylglutamine instead of formylmethionine. We further showed that coupling of the anticodon sequence change to mutations in the acceptor stem that reduced Vmax/Km(app) in formylation of the tRNAs in vitro significantly reduced their activity in initiation in vivo. In this work, we have screened an E. coli genomic DNA library in a multicopy vector carrying one of the mutant tRNA genes and have found that the gene for E. coli methionyl-tRNA synthetase (MetRS) rescues, partially, the initiation defect of the mutant tRNA. For other mutant tRNAs, we have examined the effect of overproduction of MetRS on their activities in initiation and their aminoacylation and formylation in vivo. Some but not all of the tRNA mutants can be rescued. Those that cannot be rescued are extremely poor substrates for MetRS or the formylating enzyme. Overproduction of MetRS also significantly increases the initiation activity of a tRNA mutant which can otherwise be aminoacylated with glutamine and fully formylated in vivo. We interpret these results as follows. (i) Mutant initiator tRNAs that are poor substrates for MetRS are aminoacylated in part with methionine when MetRS is overproduced. (ii) Mutant tRNAs aminoacylated with methionine are better substrates for the formylating enzyme in vivo than mutant tRNAs aminoacylated with glutamine. (iii) Mutant tRNAs carrying formylmethionine are significantly more active in initiation than those carrying formylglutamine. Consequently, a subset of mutant tRNAs which are defective in formylation and therefore inactive in initiation when they are aminoacylated with glutamine become partially active when MetRS is overproduced.
我们最近发现，大肠杆菌起始tRNA的一个突变体，其反密码子序列由CAU变为CUA，它可以利用甲酰谷氨酰胺而非甲酰甲硫氨酸从UAG起始蛋白质合成。我们还进一步发现，将反密码子序列的改变与受体茎中的突变相结合，这些突变在体外会降低tRNA甲酰化反应中的Vmax/Km(app)，这显著降低了它们在体内起始反应中的活性。在这项研究中，我们在携带其中一个突变tRNA基因的多拷贝载体中筛选了大肠杆菌基因组DNA文库，发现大肠杆菌甲硫氨酰-tRNA合成酶（MetRS）的基因部分挽救了突变tRNA的起始缺陷。对于其他突变tRNA，我们检测了MetRS过量表达对它们在起始反应中的活性以及在体内氨酰化和甲酰化的影响。部分但并非所有的tRNA突变体都能被挽救。那些无法被挽救的是MetRS或甲酰化酶的极差底物。MetRS的过量表达也显著增加了一个tRNA突变体的起始活性，否则该突变体在体内会被谷氨酰胺氨酰化并完全甲酰化。我们对这些结果的解释如下：（i）当MetRS过量表达时，作为MetRS差底物的突变起始tRNA会部分被甲硫氨酸氨酰化。（ii）在体内，被甲硫氨酸氨酰化的突变tRNA比被谷氨酰胺氨酰化的突变tRNA是甲酰化酶更好的底物。（iii）携带甲酰甲硫氨酸的突变tRNA在起始反应中的活性明显高于携带甲酰谷氨酰胺的突变tRNA。因此，当MetRS过量表达时，一部分在甲酰化方面有缺陷、因此在用谷氨酰胺氨酰化时在起始反应中无活性的突变tRNA会变得部分有活性。
