Open Accesstuf Gene Dosage Effects on the Intracellular Concentration of EF‐TuB
Author(s) -
MEIDE Peter H.,
KASTELEIN Rob A.,
VIJGENBOOM Erik,
BOSCH Leendert
Publication year - 1983
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1983.tb07167.x
Subject(s) - plasmid , microbiology and biotechnology , bacteriophage mu , transcription (linguistics) , intracellular , gene , biology , chemistry , genetics , bacteriophage , escherichia coli , linguistics , philosophy
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 (P l ) of phage λ Transcription from P l can be repressed at low temperature by a temperature‐sensitive repressor and acitvated by heat induction. Cloning occurred in two orientations in a single Eco RI site about 150 base pairs downstream of P l . Cells carrying either plasmid were shown to contain an almost doubled amount of EF‐Tu at temperatures from 28°C to 37°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°C to 43°C. The multicopy plasmid pTuB 1 described by Miyajimaet 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 .