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 Ni 21 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 EFTu 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 guaninenucleotide exchange factor EF-Ts.

The Unique tuf2 Gene from the Kirromycin Producer Streptomyces ramocissimus Encodes a Minor and Kirromycin-Sensitive Elongation Factor Tu

“…coelicolor M145 (11) was obtained from the John Innes Centre, Norwich, United Kingdom; the construction of S. coelicolor J1501 derivative LT2 is described by Olsthoorn-Tieleman et al (24). S. coelicolor strains were grown in YEME medium (11) and on R5 plates (11), when necessary supplemented with 1% (wt/vol) mannitol, 7.5 g of uracil/ml, and 50 g of histidine/ml, as described previously (11).…”

“…Chromosomal DNA from the different elfamycinproducing actinomycetes was isolated from liquid cultures grown in S medium according to the method described by Hopwood et al (11) and digested with the appropriate enzymes. Southern blotting and hybridization were performed under conditions described previously (24). The 1.0-kb MluI/NcoI, 1.0-kb NarI, and 0.65-kb SalI fragments of S. ramocissimus tuf1, tuf2, and tuf3, respectively, were used as probes after 32 P labeling by random priming (7).…”

“…S30 cell extracts of S. coelicolor LT2 (if necessary harboring pISRT2-1 or pIJ487), from which His 6 -tagged EF-Tu1 was removed by treatment with Ni 2ϩ -NTA-agarose beads, were obtained as described by Olsthoorn-Tieleman et al (24). The extracts, supplemented with purified EF-Tu species and antibiotics at various concentrations, were incubated in translation buffer (final concentrations: 50 mM Tris-HCl [pH 7.6], 9 mM MgCl 2 , 60 mM NH 4 Cl, 1 mM dithiothreitol, 1 mM ATP, 1 mM GTP, 6 mM phosphoenolpyruvate, 50 g of pyruvate kinase/ml, 0.1 mg of poly[U]/ml, 0.2 mg of E. coli tRNA/ml, and 13.2 M […”

“…Studies of the genetically well-characterized Streptomyces coelicolor revealed that this strain contains both tuf1 and tuf3 homologues (35) but lacks a tuf2 equivalent. Transcription of S. coelicolor tuf3 is subject to positive stringent control (36), and the tuf3 gene product can function as a real EF-Tu in a Streptomyces in vitro translation system (24).…”

“…In this paper we provide the sequences of the flanking genes of S. ramocissimus tuf2 and perform a transcriptional analysis of tuf2 and describe the overexpression and purification of its gene product. The actual functioning of EF-Tu2 as an EF-Tu and its interaction with kirromycin were studied by using a recently developed Streptomyces in vitro translation system (24).…”