THE TUF3 GENE OF STREPTOMYCES-COELICOLOR A3(2) ENCODES AN INESSENTIALELONGATION-FACTOR TU THAT IS APPARENTLY SUBJECT TO POSITIVE STRINGENTCONTROL

In Streptomyces coelicolor A3(2), two genes, tuf1 and tuf3, encode the apparent polypeptide chain elongation factors EF-Tu1 and EF-Tu3, resp ectively. While tuff appears to code for the major EF-Tu, the function of tuf3 is unknown. To assess the role of EF-Tu3, tuf3 was subjected to mutational and transcriptional analyses. Replacement of the 5'-half of tuf3 by an antibiotic resistance cassette had no detectable effect on phenotype, indicating that tuf3 is not essential for growth or dif ferentiation. The transcription start site of tuf3 was located approxi mately 195 nt upstream of the translation start site. The sequence of the tuf3 promoter (P-tuf3) resembles the consensus for the major class of eubacterial promoters, and P-tuf3 was recognized preferentially by an RNA polymerase fraction enriched in sigma(hrdB), the principal sig ma factor of S. coelicolor. Nuclease S1 mapping failed to reveal tuf3 transcripts during growth of S. coelicolor in liquid culture, consiste nt with the apparent absence of EF-Tu3 in total protein extracts of th e same strain. However, tuf3 transcription was observed in cultures of S. coelicolor M145 shortly after nutritional shiftdown (which resulte d in the disappearance of tuf transcripts) and after addition of serin e hydroxamate, both of which induce the stringent response. Transcript ion of tuf3 was also observed in transition-phase and stationary-phase cultures of S. coelicolor J1681, a strain deleted for bldA (which spe cifies a tRNA(leu) for the rare leucine codon UUA). In all of these ex amples, transcription of tuf3 followed the production of ppGpp, consis tent with the hypothesis that tuf3 is subject to positive stringent co ntrol.