PSEUDOURIDINE AND RIBOTHYMIDINE FORMATION IN THE TRANSFER-RNA-LIKE DOMAIN OF TURNIP YELLOW MOSAIC-VIRUS RNA

The last 82 nucleotides of the 6.3 kb genomic RNA of plant turnip yell ow mosaic virus (TYMV), the so-called 'tRNA-like' domain, presents fun ctional, structural and primary sequence homologies with canonical tRN As. In particular, one of the stem-loops resembles the T Psi(pseudouri dine)-branch of tRNA, except for the presence of a guanosine at positi on 37 (numbering is from the 3'-end) instead of the classical uridine- 55 in tRNA (numbering is from the 5'-end). Both the wildtype TYMV-RNA fragment and a;variant, TYMV-mut G(37)U in which G-37 has been replace d by U-37, have been tested as potential substrates for the yeast tRNA modification enzymes. Results indicate that two modified nucleotides were formed upon incubation of the wild-type TYMV-fragment in a yeast extract: one Psi which formed quantitatively at position 65, and one r ibothymidine (T) which formed at low level at position U-38. In the TY MV-mutant G(37)U, besides the quantitative formation of both Psi-65 an d T-38, an additional Psi was detected at position 37. Modified nucleo tides Psi-65, T-38 and Psi-37 in TYMV RNA are equivalent to Psi-27, T- 54 and Psi-55 in tRNA, respectively. Purified yeast recombinant tRNA:P si synthases (Pus1 and Pus4), which catalyze respectively the formatio n of Y-27 and Psi-55 in yeast tRNAs, are shown to catalyze the quantit ative formation of Psi-65 and Y-37, respectively, in the tRNA-like 3'- domain of mutant TYMV RNA in vitro. These results are discussed in rel ation to structural elements that are needed by the corresponding enzy mes in order to catalyze these post-transcriptional modification react ions.