A single mutation in loop IV of Escherichia coli SuIII tRNA blocks processing at both 5'- and 3'-ends of the...

We showed previously that a single mutation (T54----A54) within the highly conserved GT psi C sequence in the Escherichia coli SuIII tRNA gene results in an Su- phenotype, and we presented preliminary evidence that this mutation affected biosynthesis of the tRNA. We now show that the A54 mutation has no effect on transcription but prevents accumulation of mature mutant tRNA. The absence of mature mutant tRNA is due to blocks in processing of the precursor tRNA and is not due to instability of the mature tRNA. Characterization of the A54 precursors (approximately 130, approximately 90, and approximately 85 nucleotides long), which accumulate in E. coli minicells, indicates multiple blocks to processing of mutant transcripts. All three size classes of precursors have heterogeneous 3'-termini and contain extensions of three to seven nucleotides beyond the 3'-CCA sequence of mature tRNA indicating either a block or delay in 3'-exonucleolytic processing. The approximately 130- and approximately 90-nucleotide-long precursors also have 5'-terminal extensions suggesting a block in the 5'-processing reaction catalyzed by RNase P. Only the approximately 85-nucleotide-long precursor, which represents a small fraction of the total tRNA precursors, has the correct 5'-end of the mature tRNA.