Fragment-based lead discovery was applied to tRNA-guanine transglycosylase, an enzyme modifying post-transcriptionally tRNAs in Shigella, the causative agent of shigellosis. TGT inhibition prevents translation of Shigella's virulence factor VirF, hence reducing pathogenicity. One discovered fragment opens a transient subpocket in the preQ1-recognition site by pushing back an aspartate residue. This step is associated with reorganization of further amino acids structurally transforming a loop adjacent to the recognition site by duplicating the volume of the preQ1-recognition pocket. We synthesized 6-carboxamido-, 6-hydrazido-, and 4-guanidino-benzimidazoles to target the opened pocket, including a dihydro-imidazoquinazoline with a propyn-1-yl exit vector pointing into the transient pocket and displacing a conserved water network. MD simulations and hydration-site analysis suggest water displacement to contribute favorably to ligand binding. A cysteine residue, exclusively present in bacterial TGTs, serves as gatekeeper of the transient subpocket. It becomes accessible upon pocket opening for selective covalent attachment of electrophilic ligands in eubacterial TGTs.