16S ribosomal RNA pseudouridine synthase RsuA of Escherichia coli: Deletion, mutation of the conserved Asp102 residue, and sequence comparison among all other pseudouridine synthases

The gene for RsuA, the pseudouridine synthase that converts U516 to pseudou ridine in 16S ribosomal RNA of Escherichia coil, has been deleted in strain s MG1655 and BL21/DE3, Deletion of this gene resulted in the specific loss of pseudouridine516 in both cell lines, and replacement of the gene in tran s on a plasmid restored the pseudouridine, Therefore, rsuA is the only gene in E, coil with the ability to produce a protein capable of forming pseudo uridine516, There was no effect on the growth rate of rsuA(-) MG1655 either in rich or minimal medium at either 24, 37, or 42 degrees C, Plasmid rescu e of the BL21/DE3 rsuA- strain using pET15b containing an rsuA gene with as partate 102 replaced by asparagine or threonine demonstrated that neither m utant was active in vivo, This result supports a role for this aspartate, l ocated in a unique GRLD sequence in this gene, at the catalytic center of t he synthase, Induction of wild-type and the two mutant synthases in strain BL21/DE3 from genes in pET15b yielded a strong overexpression of all three proteins in approximately equal amounts showing that the mutations did not affect production of the protein in vivo and thus that the tack of activity was not due to a failure to produce a gene product. Aspartate102 is found in a conserved motif present in many pseudouridine synthases, The conservat ion and distribution of this motif in nature was assessed.