H-1-N-15 NMR-STUDIES OF ESCHERICHIA-COLI TRANSFER RNAPHE FROM HIST MUTANTS - A STRUCTURAL ROLE FOR PSEUDOURIDINE

Escherichia coli tRNA(U39)Phe was isolated from a specially constructed bacterial strain (DD1003/pRK3) carrying mutations in the hisT gene (the structural gene for tRNA pseudouridine synthase I) and in the pyrB gene (uracil auxotrophy). The pheU gene for tRNA(Phe) under control of the native tRNA promoter was on a multicopy plasmid and gave up to 40-fold overproduction of tRNA(U39)Phe. The double mutant permitted efficient incorporation of [3-N-15]uracil, resulting in >95% N-15 enrichment of the uracil-derived bases. H-1 and H-1-N-15 NMR experiments were used to assign the low-field proton resonances to specific hydrogen-bonding interactions. H-1 NMR assignments indicate that tRNA(U39)Phe has a structure similar to that of native tRNA(Phe) except in the anticodon region where replacement of pseudouridine (PSI) at position 39 with uridine (U) destabilizes hydrogen-bonding interactions at the base of the anticodon stem. We propose that U --> PSI modifications further stabilize interactions normally available to U by providing an additional locus for hydrogen bonding to the pyrimidine ring.