Tuf gene dosage effects on the intracellular concentration of EF-TuB.

Abstract

In this paper we have studied the effect of raising the intracellular EF-Tu concentration on the expression of tufB. To this aim cells were transformed with multicopy plasmids carrying either tufA or tufB. The intracellular EF-Tu concentrations were determined by the specific immunoelectrophoresis assay described in the preceding paper in this journal. We have cloned the tufA gene in a plasmid, containing the powerful major leftward promoter (PL) of phage lambda. Transcription from PL can be repressed at low temperature by a temperature-sensitive repressor and activated by heat induction. Cloning occurred in two orientations in a single EcoRI site about 150 base pairs downstream of PL. Cells carrying either plasmid were shown to contain an almost doubled amount of EF-Tu at temperatures from 28 degrees C to 37 degrees C. This indicates that transcription of tufA can proceed from a possible binding site for RNA polymerase on these cloned fragments. The EF-Tu level was further increased to about 30% of total cellular protein after a temperature shift from 37 degrees C to 43 degrees C. The multicopy plasmid pTuB1 described by Miyajima et al. [FEBS Lett. 102, 207-210 (1979)] and a derivative (pTuBo, compare preceding paper in this journal) were used to study the expression of both chromosomal and plasmid-borne tufB. Transformation with either plasmid raised the intracellular EF-Tu concentration by 30-60% depending on the nutritional conditions. Suppression of tufB expression was observed when the intracellular level of EF-Tu increased after transformation with all plasmids mentioned above. The results are in accord with the concept that EF-Tu acts as an autogenous feedback inhibitor involved in the regulation of tufB.