Maturation of pre-tRNA(fMet) by Escherichia coli RNase P is specified by a guanosine of the 5'-flanking sequence.

The C+1/A+72 base pair at the top of the acceptor stem of Escherichia coli tRNA(fMet) accounts for several of the specialized roles of this tRNA in translation initiation. According to the rules of RNA substrate recognition by RNase P, the C+1/A+72 pair is likely to disfavor the 5'-maturation of pre-tRNA(fMet). Indeed, in contrast to other E. coli tRNA species, tRNA(fMet) was not properly matured when overproduced from a multicopy expression vector. Half of the recovered tRNA(fMet) retained an extension at the 5' side. Such a defect of tRNA(fMet) processing could be cured by changing bases C+1 and A+72 by a Watson-Crick base pair or by non-paired bases, provided one of them was a G. It could also be compensated by either (i) over-expression of RNase P or (ii) introduction within the plasmid of one out of the three 5'-flanking sequences naturally occurring in the four E. coli tRNA(fMet) genes. The effect of these flanking sequences on the maturation of tRNA(fMet) could be accounted for by the presence of a G located 2 bases upstream from C+1. Notably, this G is the only residue that is conserved in the 5'-flanking sequences of all four E. coli tRNA(fMet) genes.