The YgjD protein is essential for the synthesis of the universal tRNA modification, N6-threonylcarbamoyladenosine (t6A), which is necessary for the decoding of ANN codons. We isolated a suppressor (ygjDsup) of the ygjDts mutant by its permissive growth at high temperature in Escherichia coli. Resequencing of the ygjDsup mutant genome showed the presence of a complicated chromosome rearrangement, an inverse insertion of a large duplicated region (c. 450 kb) into a small deleted region. The temperature-resistant growth associated with ygjDsup was due to the presence of multicopy suppressor genes, yjeE and groL, of the ygjDts mutation in the duplicated region. This DNA rearrangement was not simply mediated by IS1 transposition, but the duplicated region was flanked by IS1. We showed that the frequency of IS1 transposition was increased in ygjDts mutants. The transposase of IS1 is coded for by the insB gene, and its translation occurs through a frameshift of a ribosome translating upstream of the insA gene. We showed that this frameshifting frequency was increased by the ygjDts mutation. These results indicated that the mutation of the gene for tRNA modification, t6A, affected IS1 transposition.