The elongation factor EF-Tu from E. coli binds to the upstream activator region of the tRNA-tufB operon

Abstract

The polypeptide chain elongation factor EF-Tu of Escherichia coli is encoded by two genes, tufA and tufB, located in two different operons. Experiments in which either tufA or tufB was inactivated demonstrated that expression of the tRNA-tufB operon is dependent on a functioning tufA and thus on EF-Tu (1, to be published). In order to study a possible role of EF-Tu as trans-activator of the tRNA-tufB operon, we have investigated in vitro binding of an EF-Tu. GDP preparation to various DNA fragments of the operon. We demonstrate that specific binding occurs to a cis-acting region delimited from position -134 to the promoter, previously shown to enhance tufB transcription. Electrophoretic retardation assays reveal the formation of maximally three protein/DNA complexes, indicating that more than one protein molecule can bind to the DNA. The EF-Tu preparation used was obtained by affinity chromatography and appeared to be 95% pure. It lost its DNA binding activity upon further purification. That EF-Tu is nonetheless involved in the DNA binding is suggested by the observation that none of the three complexes is formed in the presence of kirromycin, an antibiotic that binds EF-Tu with high specificity. If so, EF-Tu.GDP most likely binds to the activator region of the tRNA-tufB operon in combination with another non-identified protein or component.