Sustenance of Escherichia coli on a single tRNAMet.

Abstract

Living organisms possess two types of tRNAs for methionine. Initiator tRNAs bind directly into the ribosomal P-site to initiate protein synthesis, and the elongators bind to the A-site during the elongation step. Eubacterial initiators (tRNAfMet) are unique in that the methionine attached to them is formylated to facilitate their binding to initiation factor 2 (IF2), and to preclude them from binding to elongation factor Tu (EFTu). However, in mammalian mitochondria, protein synthesis proceeds with a single dual function tRNAMet. Escherichia coli possesses four tRNAfMet (initiator) and two tRNAMet (elongator) genes. Free-living organisms possessing the mitochondrion like system of single tRNAMet are unknown. We characterized mutants of E. coli tRNAfMet that function both as initiators and elongators. We show that some of the tRNAfMet mutants sustain E. coli lacking all four tRNAfMet and both tRNAMet genes, providing a basis for natural occurrence of mitochondria like situation in free living organisms. The tRNA mutants show in vivo binding to both IF2 and EFTu, indicating how they carry out these otherwise mutually exclusive functions by precise regulation of their in vivo formylation. Our results provide insights into how distinct initiator and elongator methionine tRNAs might have evolved from a single ‘dual function’ tRNA.