RsuA catalyzes formation of pseudouridine at position 516 in hairpin 18 of 16S rRNA during assembly of the 30S ribosomal subunit. RsuA does not react with free unmodified 16S RNA, and only poorly with 30S particles containing unmodified RNA. The preferred substrate is an RNA fragment from residues 1 to 678 which has been complexed with 30S ribosomal proteins, suggesting that Y516 formation in vivo occurs at an intermediate stage of 30S assembly. RsuA consists of an N-terminal domain connected by an extended linker to the central and C-terminal domains. The N-terminal domain shows structural similarity to the ribosomal protein S4. RsuA is very similar in both catalytic domain structure and active site arrangement to the pseudouridine synthases RluD, TruB, and TruA, including the position of a catalytic Asp.