Elucidation of tRNA Modification Mechanism Through Structural Analysis of MnmG-tRNA Complex

Abstract

Post-transcriptional modification of wobble uridine (U34) is essential for accurate codon decoding, yet the structural principles by which the flavoprotein MnmG recognizes tRNA and positions U34 for cmnm⁵U formation have remained unresolved due to the lack of a tRNA-bound MnmG structure. In this study, we present three cryo-EM structures of Bacillus subtilis MnmG—a 2.29 Å tRNA-free structure, a 2.58 Å one-tRNA–bound structure, and a 2.51 Å two-tRNA–bound structure—that collectively establish the overall architectural framework of the enzyme in its substrate-engaged state. The two-tRNA–bound structure demonstrates that each subunit of the MnmG dimer binds one tRNA independently, revising earlier SAXS- based models that had proposed a single binding event. Furthermore, MnmE is completely dissociated in all substrate-bound states, indicating that a stable MnmE–MnmG–tRNA ternary complex is unlikely to form as a stable complex. Structural analysis reveals an A35-driven anticodon-loop reorientation mechanism, in which A35 inserts into a shallow pocket on the MnmG surface and pivots the wobble uridine U34 toward the FAD catalytic center. This local remodeling operates in concert with a broader substrate-recognition strategy in which MnmG engages conserved three-dimensional tRNA structural features—including the anticodon stem–loop, D-arm, and core body—rather than relying on base identity alone. However, the distance between U34 and the FAD isoalloxazine ring (N5–C5, ~17 Å) remains too large to permit direct methylene transfer, suggesting a potential regulatory function for the flexible 261–285 loop located between the active site and the anticodon loop. This spatial arrangement suggests that catalytic activation may require additional conformational transitions, providing an important starting point for future investigations into the temporal and regulatory aspects of U34 modification. Overall, these structures provide an atomic-level framework describing how MnmG captures and aligns tRNA for U34 modification, offering key insights that refine the structural basis of cmnm⁵U biosynthesis.