MOLECULAR RECOGNITION OF TRANSFER-RNA BY TRANSFER-RNA PSEUDOURIDINE-55 SYNTHASE

Escherichia coli tRNA pseudouridine 55 synthase catalyzes pseudouridin e formation at U55 in tRNA. A 17 base oligoribonucleotide analog of th e T-arm was equivalent to intact native tRNA as a substrate for pseudo uridine 55 synthase, viz,, the features for substrate recognition by t his enzyme are completely contained within the T-arm. The structures a nd activities of mutant tRNAs and T-arms were used to analyze substrat e recognition by pseudouridine 55 synthase. The 17-mer T-arm was an ex cellent substrate for the synthase, while disruption of the stem struc ture of the 17-mer T-arm eliminated activity. Kinetic data on tRNA mut ants lacking single T stem base pairs indicated that only the 53:61 ba se pair, which maintains the 7 base loop size, was essential for activ ity. The identities of individual bases in the stem were unimportant p rovided base pairing was intact. A major function of the T-stem appear s to be the maintainence of a stable stem-loop structure and proper pr esentation of the T-loop to pseudouridine 55 synthase. The 7 base T-lo op could be expanded or contracted by 1 base and still retain activity , albeit with a 30-fold reduction in k(cat), Kinetic analysis of T-loo p mutants revealed the requirement for U54, U55, and A58, and a prefer ence for C over U at position 56. Base substitutions at loop nonconser ved position 59 or semiconserved positions 57 or 60 were well tolerate d. Comparison of pseudouridine 55 synthase and tRNA (m(5)U54)-methyltr ansferase revealed that both enzymes required the stem-loop structure. However, pseudouridine 55 synthase was not stringent for a 7 base loo p and recognized a consensus base sequence within the T-loop, while tR NA (m(5)U54)-methyltransferase recognized the secondary structure of t he 7 member T-loop with only a specific requirement for U-54, the T-lo op substrate site, We conclude that recognition of tRNA by pseudouridi ne 55 synthase resides in the conformation of the T-arm plus four spec ific bases of the loop.