The variant tuf3 gene of Streptomyces coelicolor A3(2) encodes a real elongation factor Tu, as shown in a novel Streptomyces in vitro translation system.

Abstract

In Streptomyces coelicolor, the regular and abundant elongation factor (EF)-Tu1 is encoded by tuf1, while the actual function of the highly divergent tuf3 gene product is not yet known. Expression of the latter could so far only be detected on the transcriptional level under stress conditions. In this paper we demonstrate the presence of low levels of EF-Tu3 in strains of the J1501 lineage. Enhanced expression was observed for J1501 glkA and relA deletion mutants, which lack glucose kinase and ribosome-bound ppGpp synthetase, respectively. To assess the putative translational capacities of EF-Tu3, a novel Streptomyces in vitro translation assay was designed, based on the full elimination by Ni2+ affinity adsorption of chromosomally encoded (His)6-tagged EF-Tu1 from a S. coelicolor cell-free extract. Translational activity of this system is totally dependent on the addition of purified EF-Tu species or on the presence of an additional elongation factor Tu in the extract, e.g. encoded by a plasmid-borne tuf gene. Using this EF-Tu-dependent translation system, we have established that S. coelicolor EF-Tu3 has translational capacities despite its striking deviation from the common prokaryotic EF-Tu sequence at positions involved in either aminoacyl-tRNA binding or interaction with the guanine-nucleotide exchange factor EF-Ts.