mocimycin and Chromosome-Deletion

Of the two plasmids pTUB1 and pTUB2 constructed by cloning of the 8.9 kb EcoRI fragment carrying tufB (Miyajima, A., Shibuya, M., & Kaziro, Y. (1979) FEBS Lett. 102, 207-210), pTUB2 possesses a deletion of about 0.3 kb. Restriction and sequence analyses have located the deletion in the region of the four tRNA genes thrU-tyrU-glyT-thrT upstream of the tufB structural gene. As a result of homologous recombination between thrU and thrT, the four tRNA genes have been replaced by a single thrU-thrT hybrid gene. The deletion of the three tRNA genes does not significantly alter the in vivo expression of tufB as assessed by the kirromycin-sensitive phenotype of the transformant cells and by the synthesis of EF-Tu in mini-cells. Nor does the deletion affect the synthesis of beta-galactosidase in lysogens carrying a lambda transducing phage with a tufB-lacZ fusion. Transcription of tufB and synthesis of EF-Tu in a cell-free transcription-translation coupled system were essentially the same, regardless of whether pTUB1 or pTUB2 DNA was used as a template. Likewise, 0.2 mM ppGpp inhibits the synthesis of tufB mRNA on both pTUB1 and pTUB2 templates to the same extent. We concluded that the replacement by thrU-thrT hybrid gene of the four tRNA genes upstream of the tufB coding region does not significantly affect either in vivo or in vitro expression of tufB.

Topics: Bacteriophage lambda; Base Sequence; Cell-Free System; Chromosome Deletion; Drug Resistance, Microbial; Escherichia coli; Mutation; Peptide Elongation Factor Tu; Peptide Elongation Factors; Plasmids; Pyridones; RNA, Bacterial; RNA, Transfer