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The unique leucine-, arginine-, valine-, and phenylalanine-specific transfer ribonucleic acids (tRNA's) produced in relaxed-control (rel-) Escherichia coli during leucine or arginine starvation are chromatographically similar to those produced by chloramphenicol treatment. The major unique rel- leucine-specific and phenylalanine-specific tRNA's are heterogeneous, accumulate with time of starvation, and can account for up to 70% of the respective amino acid acceptor activities. The changes which occur in the isoacceptor profiles for tRNALeu and tRNAPhe as a function of starvation time suggest that the unique species are undermodified precursors to the major isoacceptor species observed in nonstarved cells. Analyses of the isoacceptor patterns of tRNA from cells recovering from starvation suggest that the unique species of tRNALeu and tRNAPhe may not be normally occurring precursors. When leucine-starved cells were incubated in fresh, fully supplemented medium, the major unique tRNALeu and tRNAPhe appeared to be converted to normal species only slowly or not at all. The results are consistent with the view that some of the events in the post-transcriptional modification of tRNA may occur in an ordered sequence. An examination of the subcellular distribution of the unique leucine and phenylalanine tRNA's revealed that these species occur on the ribosome at about the same frequency as the major, normally occurring isoacceptor species. This result provides additional evidence of a precursor-product relationship for the unique and normal tRNA's and further indicates that there is no discrimination against the unique species by the ribosome.

The structures of the major, chromatographically unique phenylalanine and leucine tRNAs produced during leucine starvation of a relaxed control (rel-) mutant of E. coli have been determined. The results demonstrate that the unique species are 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-(delta2-isopentenyl)adenosine-37, a mixture of U and pseudouridine (psi) in position 39, a mixture of U and 3-(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 psi55, indicating that that species is a mixture of mature and undermodified tRNAs. The unique tRNALeu isoacceptor consists of two 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; one subspecies also lacks D17. Compared with the tRNALeusI from wild-type strains of E. coli B and K12, both tRNALeuI from nonstarved cells and the unique, rel-tRNALeu are deficient in the modified guanosine which normally occurs adjacent to the anticodon and the pseudouridine in the GTpsiC sequence of the psi loop. Both the unique tRNAPhe and the unique tRNALeu lack dihydrouridine residues which occur in the 5' half of the D loop and pseudouridines which occur in the 3' half of the anticodon loop and adjoining stem. Taken together, these findings suggest that the same enzymes are responsible for the formation of these particular modified bases in both tRNAs. The results further suggest that several, perhaps most, of the tRNAs from cells cultured under conditions in which RNA and protein synthesis are uncoupled will be similarly deficient in dihydrouridine and pseudouridine and other minor nucleosides which occur less frequently. Because both modification-deficient rel-tRNAs have dihydrouridine at position 20 and pseudouridine in the psi loop (and at position 41 in the unique tRNALeu), the results support the view that there was 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 posttranscriptional modification of newly synthesized tRNA.

Inhibition of protein synthesis in relaxed control E. coli results in the formation of chromatographically unique isoacceptor species of phenylalanine tRNA. The genetic origin and some functional properties of the major unique species of tRNA (Phe) produced during leucine starvation were investigated. RNA:DNA hybridization analyses revealed that the normally occurring and major unique species of tRNA (Phe) are generated from DNA sequences which are identical or closely related and that there may be only one such sequence in the E. coli chromosome. Results from 32P pulse-chase experiments revealed that the unique tRNA (Phe) can be converted to a chromatographically normal form upon resumption of cell growth in fully supplemented medium. These findings, taken with earlier results which indicate that the unique species is not derived from preexisting, normally occurring species, indicate that the unique tRNA(Phe) is a modification-deficient form of the normal species. Comparative studies of the unique and normal phenylalanine tRNAs revealed that the unique species is aminoacylated at a much lower rate than the normal species and is only about 60% as efficient in a tRNA-dependent, poly(U)-directed protein synthesizing system.

The heterogeneity of undermodified phenylalanine tRNA produced in relaxed control E. coli during amino acid starvation was investigated. Examination of the RPC-5 elution profiles of tRNAPhe prepared from non-starved cells and cells starved of a variety of amino acids, including some known to be involved in the formation of modified bases revealed that: (1) only one species of fully modified tRNAPhe appears to occur in cells grown in enriched medium; (2) at least two chromatographically unique isoacceptor species are observed in addition to the normal tRNAPhe in starved cells; (3) the unique, undermodified species of tRNAPhe from leucine-starved cells, known to be deficient in dihydrouridine, pseudouridine, 2-thiomethyl-N6-(delta2-isopentenyl) adenosine and 3-(3-amino-3-carboxypropyl) uridine, co-elute with the unique species produced in cells starved of histidine or arginine or treated with puromycin or chloramphenicol; (4) additional unique species of tRNAPhe can be detected in methyl- and sulfur-deficient tRNA from methionine- and cysteine-starved cells; (5) analysis of phenoxyacetylated tRNA revealed that the chromatographically unique and normal species from starved cells contain subspecies deficient in 3-(3-amino-3-carboxypropyl) uridine; and (6) using phenoxyacetylation as a means of effecting the resolution of undermodified subspecies, a total of at least ten chromatographically unique subspecies of rRNAPhe were detected in an organism that appears to posses only one gene for tRNAPhe. Taken together, the results support the view that there are both general and specific effects of amino acid starvation on the post-transcriptional modification of tRNA.