The thiolation of U34 at carbon 2 in tRNA by Escherichia coli MnmA precedes modification at carbon 5 and is dependent on a [4Fe-4S] cluster.

Chemical modifications of transfer RNAs (tRNAs) fine-tune the recognition of messenger RNA codons on the ribosome for accurate protein translation. Uridine 34 (U34) of the anticodon of three tRNAs is modified in all domains of life. In bacteria, U34 is modified at C5 by aminomethyl derivatives by MnmE/MnmG enzymes and at C2 by a sulfur atom by MnmA enzymes from two distinct classes. C-type MnmAs possess a CXXC+ C catalytic motif, in which cysteines bind a [4Fe-4S] cluster essential for catalysis. D-type MnmAs possess a DXXC+ C catalytic motif, which can also bind a [4Fe-4S] cluster, but the function of the cluster is controversial. To shed light on the role of the [4Fe-4S] cluster in the thiolation reaction, we report here new in vitro catalytic assays of the D-type Escherichia coli MnmA enzyme (EcMnmA) before, and after cluster reconstitution under anaerobic conditions. We confirm that that only holo-EcMnmA can thiolate U34 in a tRNA transcript using sulfide as a sulfur source and show that 1) a bulk E. coli ΔmnmA tRNA, containing the C5 modification, is not a substrate, indicating that thiolation at C2 precedes modification at C5, 2) cysteine with IscS cannot provide the sulfur atom for tRNA thiolation without a reductant and 3) the variant, in which the aspartate of the DXXC + C motif is replaced by cysteine, is efficient in U34-tRNA thiolation in vitro but not in vivo, raising questions about the in vivo function of soft cluster coordination.