Varshney U, Lee C P, RajBhandary U L
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2305-9. doi: 10.1073/pnas.90.6.2305.
We show that the two most important properties needed for a tRNA to function in initiation in Escherichia coli are its ability to be formylated and its ability to bind to the ribosomal P site. This conclusion is based on conversion of two different elongator tRNAs to ones that can act as initiators in E. coli. We transplanted the features unique to E. coli and eubacterial initiator tRNAs to E. coli elongator methionine tRNA (tRNA(Met)) along with an anticodon sequence change and analyzed their activities in initiation in E. coli. Introduction of a C1.A72 mismatch at the end of the acceptor stem of tRNA(Met), which generates the minimal features necessary for formylation, produces a tRNA with very low activity in initiation. Subsequent introduction of three consecutive G.C base pairs at the bottom of the anticodon stem, which is necessary for ribosomal P site binding, produces a tRNA with significant activity in initiation. Furthermore, introduction of the features necessary for formylation and for ribosomal P site binding into E. coli elongator glutamine tRNA produces a tRNA that initiates protein synthesis in E. coli.
我们发现，在大肠杆菌中，tRNA要在起始过程中发挥作用，需要具备的两个最重要特性是其被甲酰化的能力以及与核糖体P位点结合的能力。这一结论基于将两种不同的延伸tRNA转化为可在大肠杆菌中充当起始tRNA的过程。我们将大肠杆菌和真细菌起始tRNA特有的特征与反密码子序列变化一起移植到大肠杆菌延伸甲硫氨酸tRNA（tRNA(Met)）上，并分析了它们在大肠杆菌起始过程中的活性。在tRNA(Met)受体茎末端引入C1.A72错配，这产生了甲酰化所需的最小特征，产生了一种在起始过程中活性非常低的tRNA。随后在反密码子茎底部引入三个连续的G.C碱基对，这是核糖体P位点结合所必需的，产生了一种在起始过程中具有显著活性的tRNA。此外，将甲酰化和核糖体P位点结合所需的特征引入大肠杆菌延伸谷氨酰胺tRNA中，产生了一种能在大肠杆菌中起始蛋白质合成的tRNA。
