J. Mol. Biol. (1977) 117, 1061-1079 Cysteine Transfer RNA of Escherichia coli: Nucleotide Sequence and Unusual Metabolic Properties of the 3' C-C-A Terminus GAlL P. MAZZARA AND WILLIAM H. McCLAIN Department of Bacteriology, University of Wisconsin, Madison, Wise. 53706, U.S.A. (Received 16 September 1977) Transfer RNA nucleotidyltransferase is required for the normal growth of Escherichia cell and for biosynthesis of some bacteriophage tRNAs. In order to investigate the involvement of this enzyme in the biosynthesis of E. cell tRNA we have studied the synthesis of these molecules in wild-type (cea+) and tRNA nucleotidyltransferase-deficient (cca) strains. We found that a small subset of tRNA species lack a complete C-C-A terminus in the cea strain. Within this subset of tRNAs, tt~NA cy~ was the most severely affected; nucleotide sequence analysis showed that 79% of tRNA cy~ molecules lacked one or more of the 3'-terminM C-C-A residues. However, in this same cca strain, the tRNA cy~ synthesized in the presence of chloramphenico], an antibiotic which inhibits turnover of the terminal AMP residue of tRNAs, possessed a complete C-C-A terminus. These results indicate that tRNA nucleotidyltransferase is not required for the bio- synthesis of tRNA cy~. We infer from these findings that the 3' C-C-A sequence of tRIqACys arises during transcription, and that the high proportion of defective tRNA cy~ molecules found in eca cells results from unusually high turnover at the 3' terminus of this tRNA. This conclusion must be stated with some reservation, for it is based on studies using a mutan~ strain which contains 2 % residual tRNA nucleotidyltransferase activity. Regardless of its mode of biosynthesis, however, tRNA cys does possess an unusual metabolic feature in that it is subject to rapid end-turnover. We have not determined why tRNA cy~ exhibits such unusual behavior. 1. Introduction Transfer RNA molecules are transcribed as large precursor RNAs which are enzymatically processed in an ordered series of steps to generate mature tRNAs (see McClain, 1977). Investigators are presently attempting to elucidate the path- ways by which precursor RNAs are converted into tRNAs; such studies have included characterization of biosynthetic intermediates, as well as identification of the participating enzymes. One aspect of tRNA biosynthesis currently under investigation is the origin of the 3' terminus of tI~NAs. All tRNA molecules have the nucleotide sequence C-C-A a~ the 3' terminus; this sequence is required for the accepter and ~ransfer functions of these molecules. Early studies of tRNA identified an enzyme, tRNA nucleotidyl. transferase (E.C. 2.7.7.25), capable of incorporating AMP and CMP residues into tRNA molecules lacking all or part of the C-C-A terminus (see Deutscher, 1973). Thus, the ubiquity of the 3'-terminal C-C-A sequence and the existence of an enzyme 1061