Modification-deficient transfer ribonucleic acids from relaxed control Escherichia coli: structures of the major undermodified phenylalanine and leucine transfer RNAs produced during leucine starvation

Abstract

The structures of the major, chromatographically unique phenylalanine and leucine tRNA produced during leucine starvation of a relaxed control (rel-) mutant of E. coli were determined. The unique species are apparently modification-deficient forms of the major, normally occurring isoacceptor species. The unique tRNAPhe differs from the fully modified species at nucleotide positions 16, 37, 39, 47 and 55 from the 5'' terminus. The unique species contains uridine (U) in place of dihydrouridine-16 (D16), isopentenyladenosine in place of 2-thiomethyl-N6-(.delta.2-isopentenyl)adenosine-37, a mixture of U and pseudouridine (.psi.) in position 39, a mixture of U and 3-(3-amino-3-carboxypropyl)uridine at position 47, and a mixture of U and .psi. at position 55. The chromatographically normal isoacceptor from amino acid starved cells is deficient in D16 and .psi.55, indicating that the species is a mixture of mature and undermodified tRNA. The unique tRNALeu isoacceptor consists of 2 subspecies which are undermodified forms of the major, normally occurring isoacceptor, tRNALeuI. Both unique subspecies lack the D and .psi. residues which occur at positions 16 and 39 from the 5'' terminus; 1 subspecies also lacks D17. Compared with the tRNALeuI from wild-type strains of E. coli B and K12, tRNALeuI from nonstarved cells and the unique, rel- tRNALeu are deficient in the modified guanosine which normally occurs adjacent to the anticodon and the .psi. in the GT.psi.C sequence of the .psi. loop. The unique tRNAPhe and the unique tRNALeu lack D residues which occur in the 5'' half of the D loop and .psi. which occur in the 3'' half of the anticodon loop and adjoining stem. The same enzymes are apparently responsible for the formation of these particular modified bases in both tRNA. Several, perhaps most, of the tRNA from cells cultured under conditions in which RNA and protein synthesis are uncoupled probably will be similarly deficient in D and .psi. and other minor nucleosides which occur less frequently. Because both modification-deficient rel- tRNA have D at position 20 and .psi. in the .psi. loop (and at position 41 in the unique tRNALeu), there are apparently multiple D- and .psi.-forming enzymes in E. coli, some of which may turn over rapidly or are selectively inactivated when protein synthesis is blocked. The results are discussed with a view toward understanding the structural basis for the altered biological activity of the unique tRNAPhe species and the order of events in the post-transcriptional modification of newly synthesized tRNA.