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Evidence that a single EF-Ts suffices for the recycling of multiple and divergent EF-Tu species in Streptomyces coelicolor A3(2) and Streptomyces ramocissimus

Department of Biochemistry, Leiden Institute of Chemistry, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands¹

Author for correspondence: Barend Kraal. Tel: +31 71 5274770. Fax: +31 71 5274340. e-mail: B.kraal{at}chem.leidenuniv.nl

The tsf genes from Streptomyces coelicolor A3(2) and Streptomyces ramocissimus, encoding the guanine-nucleotide exchange factor EF-Ts, were cloned and sequenced. Streptomycetes have multiple and highly divergent EF-Tu species, with EF-Tu1 and EF-Tu3 showing only about 65% amino acid sequence identity, and yet these can apparently interact with a single EF-Ts species. tsf lies in an operon with rpsB, which encodes ribosomal protein S2. The amino acid sequence of S2 from S. coelicolor differs from most other bacterial S2 homologues in having a C-terminal extension of 70 aa residues with a highly repetitive organization, the function of which is unknown. Transcription analysis of the rpsB–tsf operon of S. coelicolor by promoter probing, nuclease S1 mapping and Northern blotting revealed that the genes give rise to a bicistronic transcript from a single promoter upstream of rpsB. An attenuator was identified in the rpsB–tsf intergenic region; it results in an approximately 2:1 ratio of rpsB vs tsf transcripts. Although tuf1, encoding the major EF-Tu, is located in the rpsL ribosomal protein operon, an additional promoter in the fus–tuf1 intergenic region leads to a significant excess of EF-Tu over ribosomes. Most amino acid residues known from the Escherichia coli crystal structure of the EF-Tu·EF-Ts complex to be directly involved in interaction between the two elongation factors are conserved between E. coli and Streptomyces. However, whenever interaction residues in the EF-Tu moiety show divergence among Streptomyces EF-Tu1, EF-Tu2 and EF-Tu3, the single Streptomyces EF-Ts exhibits compensatory substitutions of the corresponding residues. These apparently enable productive interaction to occur with all three EF-Tus.

Keywords: rpsB operon, tsf regulation, GTPase switch protein, guanine-nucleotide exchange factor, ribosomal protein S2

Abbreviations: EF, elongation factor

The GenBank accession numbers for the sequences of the S. coelicolor rpsB–tsf operon and the S. ramocissimus tsf gene determined in this work are AF034101 and AF130345, respectively.

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