TRANSFER-RNA RECOGNITION BY THE ESCHERICHIA-COLI DELTA(2)-ISOPENTENYL-PYROPHOSPHATE-TRANSFER-RNA DELTA-2-ISOPENTENYL TRANSFERASE - DEPENDENCE ON THE ANTICODON ARM STRUCTURE

To elucidate the sequence elements required in the anticodon stem for the recognition of Escherichia coli tRNA(Ser) (GGA) by the E. coli iso pentenyl-tRNA:A(37) transferase (IPTT), which result in the conversion of A(37) into isopentenylated i(6)A(37) we have tested and characteri zed in vitro T7-runoff transcripts of 17 variants of E. coli tRNA(Ser) (GGA) and 7 other tRNAs from E. coli and yeast. Our results indicate t hat, instead of a stringent specific anticodon stem and loop sequence, the key feature required for the recognition of E. coli tRNAs by IPTT is the A(36)A(37)A(38) sequence occurring within the seven-membered a nticodon loop, and the retention of the standard helical structure and flexibility, especially in the proximal anticodon stem. The G(30)U-4 0 mismatch base pair close to the anticodon loop is strictly avoided. The frequent occurrence of a C-G base pair in the three stem locations closest to the loop (positions 29-41, 30-40 and 31-39) or the occurre nce of even one such C-G base pair along with some other similarly les s suited, but individually tolerated deviations can also totally aboli sh the A(37) isopentenylation of tRNA. For the position 30-40, the G-C base pair is shown uniquely suited, whereas for the adjoining 29-41 s tem location, a purine-pyrimidine base pair with pyrimidine on the 3'- side is strongly preferred. Retention of the overall 3D tRNA structure is favorable for isopentenylation and allows some tolerance of proxim al stem sequence deviations. Our data suggest a recognition mode that implies the interaction of IPTT with the strictly conserved A(36)A(37) A(38) sequence and the other functional groups located in the minor gr oove of the anticodon stem.