Importance of formylability and anticodon stem sequence to give a tRNA(Met) an initiator identity in Escherichia coli.

In bacteria, the free amino group of the methionylated initiator tRNA is specifically modified by the addition of a formyl group. The functional relevance of such a formylation for the initiation of translation is not yet precisely understood. Advantage was taken here of the availability of the fmt gene, encoding the Escherichia coli Met-tRNA(fMet) formyltransferase, to measure the influence of variations in the level of formyltransferase activity on the involvement of various mutant tRNA(fMet) and tRNA(mMet) species in either initiation or elongation in vivo.

Critical role of the acceptor stem of tRNAs(Met) in their aminoacylation by Escherichia coli methionyl-tRNA synthetase.

To be aminoacylated by Escherichia coli methionyl-tRNA synthetase, a tRNA requires the presence of the methionine anticodon (CAU sequence). However, the importance in this reaction of the other nucleotides of tRNAs(Met) has still to be described. In this work, through the study of more than 35 variants of tRNAs(Met), it is shown, firstly, that the parameters of the aminoacylation reaction remain independent of the mutations affecting either the sequences or the sizes of the D-loop, D-stem and variable loop.

Involvement of the size and sequence of the anticodon loop in tRNA recognition by mammalian and E. coli methionyl-tRNA synthetases.

The rates of the cross-aminoacylation reactions of tRNAs(Met) catalyzed by methionyl-tRNA synthetases from various organisms suggest the occurrence of two types of tRNA(Met)/methionyl-tRNA synthetase systems. In this study, the tRNA determinants recognized by mammalian or E. coli methionyl-tRNA synthetases, which are representative members of the two types, have been examined.

Disruption of the gene for Met-tRNA(fMet) formyltransferase severely impairs growth of Escherichia coli.

In bacteria, as well as in chloroplasts and mitochondria, the free amino group of the methionylated initiator tRNA(fMet) is specifically modified by the addition of a formyl group. The importance of this modification remains unclear. With the availability of pure Escherichia coli 10-formyltetrahydrofolate:L-methionyl-tRNA(fMet) N-formyltransferase, the enzyme catalyzing Met-tRNA(fMet) formylation, the corresponding fmt gene and its flanking regions were cloned and sequenced.

Nucleotides of tRNA governing the specificity of Escherichia coli methionyl-tRNA(fMet) formyltransferase.

In Escherichia coli, the free amino group of the aminoacyl moiety of methionyl-tRNA(fMet) is specifically modified by a transformylation reaction. To identify the nucleotides governing the recognition of the tRNA substrate by the formylase, initiator tRNA(fMet) was changed into an elongator tRNA with the help of an in vivo selection method. All the mutations isolated were in the tRNA acceptor arm, at positions 72 and 73.