The expression of the cloned tufB gene in vivo

In [I], we have reported the construction and characterization of two hybrid ColEl plasmids, pTUB1 and pTUB2, in which the 8.9 kilobases (kb) (18.6% h-unit) EcoRI fragment derived from transducing phage XrifdlS was inserted in two different orientations into a ColEl derivative plasmid RSF2124. The 8.9 kb fragment contained a part of rrnB, genes for 4 tRNAs (glyT, tyrU, thrT, and thrum), tujB, a gene for an unidentified protein ‘U’, and a part of rplK [2] (fig.1). In a cell-free transcription-translation system, both pTUB1 and pTUB2 DNA could direct the synthesis of tufB mRNA and EF-Tu(B) (product of &@) [l]. Furthermore, the analysis of the transcripts synthesized in the presence of purified RNA polymerase holoenzyme has revealed that the transcription was initiated at a point -300 nucleotides upstream of the structural gene for tujB (Shibuya, A. M., Y. K., in preparation) and was specifically inhibited by low concentrations of ppGpp (A. M., Shibuya, Y. K., in preparation). These results suggests that the promotor gene for tuj73 which is under the stringent control may be located in close proximity to its structural gene. Here, we have studied the expression of the cloned tujB gene within cells of the kirromycin-resistant mutant LBE2012 [3,4]. The antibiotic kirromycin inhibits protein synthesis by preventing the release of EF-Tu from ribosomes [S--7]. The mutant LBE2012 was originally thought to have the tufA mutant allele coding for a functional kirromycin-resistant EF-Tu(A) and the mutant tujB gene that produces a non-functional EF-Tu(B) [3,4]. However, the tufB product of LBE2012 is functional in the absence of kirromycin, but is inactivated in the presence of kirromycin (L.