Pseudouridine is the most abundant of more than 100 chemically distinct natural 
ribonucleotide modifications. Its synthesis consists of an isomerization 
reaction of a uridine residue in the RNA chain and is catalyzed by pseudouridine 
synthases. The unusual reaction mechanism has become the object of renewed 
research effort, frequently involving replacement of the substrate uridines with 
5-fluorouracil (f(5)U). f(5)U is known to be a potent inhibitor of pseudouridine 
synthase activity, but the effect varies among the target pseudouridine 
synthases. Derivatives of f(5)U have previously been detected, which are thought 
to be either hydrolysis products of covalent enzyme-RNA adducts, or 
isomerization intermediates. Here we describe the interaction of pseudouridine 
synthase 1 (Pus1p) with f(5)U-containing tRNA. The interaction described is 
specific to Pus1p and position 27 in the tRNA anticodon stem, but the enzyme 
neither forms a covalent adduct nor stalls at a previously identified reaction 
intermediate of f(5)U. The f(5)U27 residue, as analyzed by a DNAzyme-based assay 
using TLC and mass spectrometry, displayed physicochemical properties unaltered 
by the reversible interaction with Pus1p. Thus, Pus1p binds an f(5)U-containing 
substrate, but, in contrast to other pseudouridine synthases, leaves the 
chemical structure of f(5)U unchanged. The specific, but nonproductive, 
interaction demonstrated here thus constitutes an intermediate of Pus turnover, 
stalled by the presence of f(5)U in an early state of catalysis. Observation of 
the interaction of Pus1p with fluorescence-labeled tRNA by a real-time readout 
of fluorescence anisotropy and FRET revealed significant structural distortion 
of f(5)U-tRNA structure in the stalled intermediate state of pseudouridine 
catalysis.