Post-Transcriptional Modifications of the Anticodon Loop Region: Alterations in Isoaccepting Species of tRNA's During Development in Bacillus subtilis

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Abstract
Structural similarities of tRNA9s were compared using three sets of isoaccepting species that had previously been shown to undergo significant changes in chromatographic elution properties as a function of developmental stage in Bacillus subtilis. Comparisons of the structures of the tRNA9s were based on the composition of their modified nucleosides, comparisons of oligonucleotide elution profiles from RPC-5 columns, and two-dimensional electrophoretic fingerprint analysis of oligonucleotides. The tRNA9s studied were tRNALys1 and tRNALys3; tRNATyr1 and tRNATyr2; and tRNATrp1 and tRNATrp2. The results suggest that the difference among these pairs of isoaccepting species is a difference in the degree of post-transcriptional modifications of the anticodon loop region. The nucleosides involved were N6-(Δ2-isopentenyl)adenosine (i6A), 2-methylthio-N6-(Δ2-isopentenyl)adenosine (ms2i6A), and an unknown nucleoside K, which occurred in a position analogous to N-[9-(β-d-ribofuranosyl)purin-6-ylcarbamoyl]threonine. The amounts of i6A and ms2i6A, determined using total tRNA from exponential-or stationary-phase cells, suggest that the thiomethylation of i6A is a pleiotropic phenomenon affecting several tRNA species. As opposed to the situation in Escherichia coli tRNA, where ms2i6A constitutes about 90% of the total hydrophobic nucleosides at all growth stages, B. subtilis tRNA9s have i6A as the predominant hydrophobic nucleoside in exponential growth and ms2i6A as the predominant nucleoside in stationary phase. Thus, the enzyme system which forms i6A and the enzyme system which thiomethylates i6A are not coordinated during growth in B. subtilis as they are in E. coli. It is suggested that these changes in anticodon loop modifications in B. subtilis may be related to changes in the translational apparatus which occur during sporulation. Images