Published on March 1, 2012 in Nucleic Acids Res volume 40.
PubMed ID: 22102571
Pseudouridine synthase 1 (Pus1p) is an unusual site-specific modification enzyme in that it can modify a number of positions in tRNAs and can recognize several other types of RNA. No consensus recognition sequence or structure has been identified for Pus1p. Human Pus1p was used to determine which structural or sequence elements of human tRNA(Ser) are necessary for pseudouridine (Psi) formation at position 28 in the anticodon stem-loop (ASL). Some point mutations in the ASL stem of tRNA(Ser) had significant effects on the levels of modification and compensatory mutation, to reform the base pair, restored a wild-type level of Psi formation. Deletion analysis showed that the tRNA(Ser) TPsiC stem-loop was a determinant for modification in the ASL. A mini-substrate composed of the ASL and TPsiC stem-loop exhibited significant Psi formation at position 28 and a number of mutants were tested. Substantial base pairing in the ASL stem (3 out of 5 bp) is required, but the sequence of the TPsiC loop is not required for modification. When all nucleotides in the ASL stem other than U28 were changed in a single mutant, but base pairing was retained, a near wild-type level of modification was observed.