Published on Nov. 1, 2013 in Nucleic Acids Res volume None.

PubMed ID: 24293650


Abstract:

In Escherichia coli, the MnmEG complex modifies transfer RNAs (tRNAs) decoding NNA/NNG codons. MnmEG catalyzes two different modification reactions, which add an aminomethyl (nm) or carboxymethylaminomethyl (cmnm) group to position 5 of the anticodon wobble uridine using ammonium or glycine, respectively. In $${\hbox{ tRNA }}_{\hbox{ cmnm5s2UUG }}^{\hbox{ Gln }}$$ and $${\hbox{ tRNA }}_{\hbox{ cmnm5UmAA }}^{\hbox{ Leu }}$$, however, cmnm5 appears as the final modification, whereas in the remaining tRNAs, the MnmEG products are converted into 5-methylaminomethyl (mnm5) through the two-domain, bi-functional enzyme MnmC. MnmC(o) transforms cmnm5 into nm5, whereas MnmC(m) converts nm5 into mnm5, thus producing an atypical network of modification pathways. We investigate the activities and tRNA specificity of MnmEG and the MnmC domains, the ability of tRNAs to follow the ammonium or glycine pathway and the effect of mnmC mutations on growth. We demonstrate that the two MnmC domains function independently of each other and that $${\hbox{ tRNA }}_{\hbox{ cmnm5s2UUG }}^{\hbox{ Gln }}$$ and $${\hbox{ tRNA }}_{\hbox{ cmnm5UmAA }}^{\hbox{ Leu }}$$ are substrates for MnmC(m), but not MnmC(o). Synthesis of mnm5s2U by MnmEG-MnmC in vivo avoids build-up of intermediates in $${\hbox{ tRNA }}_{\hbox{ mnm5s2UUU }}^{\hbox{ Lys }}$$. We also show that MnmEG can modify all the tRNAs via the ammonium pathway. Strikingly, the net output of the MnmEG pathways in vivo depends on growth conditions and tRNA species. Loss of any MnmC activity has a biological cost under specific conditions.


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