From elongator tRNA to initiator tRNA

Abstract

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.