RluF modifies U2604 in a stem-loop (helix 93 of the Peptidyl transferase Center on Domain V) of 23S RNA, while its homologue, RluB, modifies the adjacent base, U2605. Both uridines are in the same RNA stem, separated by only 4 A. The RNA stem-loop is bound to a conserved binding groove in the catalytic domain. A base from a bulge in the stem, A2602, folds into the stem, forcing one strand of the RNA stem to translate by one position and thus positioning U2604 to flip into the active site. The structure of the catalytic domain of RluF is similar to those of RsuA and TruB. RluF is a member of the RsuA family of pseudouridine synthases, along with RluB and RluE (but not TruB), present in all kingdoms of life.