~The regulatory functions of the leader region preceding the Lactococcus lactis trp operon have been studied by mutagenesis analysis. This leader presents striking similarity to 'T-box' leaders found upstream of many Gram-positive aminoacyl-tRNA synthetase genes and some amino acid biosynthesis operons, which are controlled by antitermination through interaction of the leader transcript with cognate uncharged tRNA. A region of the L. lactis leader transcript also contains a series of (GN)AG repeats which, in Bacillus, are involved in the binding of the trp RNA-binding protein (TRAP) which controls trp transcription. A screen was developed for the isolation of regulatory mutants affected in the leader region. All spontaneous mutants contained deletions; point mutations were only obtained after UV-induced mutagenesis. All mutations affected the putative transcription terminator upstream of the trp operon, demonstrating that trp is indeed controlled by transcription antitermination. Keywords INTRODUCTIONAll seven genes required for tryptophan (Trp) biosynthesis form an operon in Lactococcus lactis (Bardowski et al., 1992), which is preceded by a 403-base non-coding leader region (Fig. 1). In the presence of Trp, two small transcripts are produced, corresponding to parts of the leader region : a 290-base transcript, initiated at the Ptrp transcription promoter and arrested close to the putative transcription terminator T1 upstream of the structural genes, and a 160-base transcript, identical to the 3' part of the former, corresponding to a processing product (Raya et al., 1998). Under conditions of Trp starvation, transcription of a third additional transcript is induced 300-to 500-fold. This 8 kb transcript spans the full length of the operon.The trp leader region presents striking structural similarities to a family of leader regions upstream of t Present address: IBB-PAN, Zaklad Biochemii Drobnoustrojow, UI.Pawinskiego SA, 02-106 Warszawa, Poland.Abbreviations: TRAP, trp RNA-binding protein; 6-fTrp, 6-fluorotryptophan.aminoacyl-tRNA synthetase genes and some amino acid biosynthesis operons in a number of Gram-positive bacteria, which are controlled by antitermination . All these leader regions contain a transcription terminator upstream of the coding sequence. The sequence immediately upstream of the terminator, and extending within its 5' part, has the potential to form an alternative, but much less stable, antiterminator structure, in which a 14-base region, called the T-box, is highly conserved. The leader transcripts also have in common conserved stem-loop structures, a single codon, specifying the amino acid expected to regulate expression of the system, and an antiacceptor, complementary to the tRNA amino acid acceptor arm, present within the T-box (Fig. 1). The conservation of these features suggested a regulatory model in which transcription is controlled by a switch between the antiterminator and the terminator form of the leader transcript, mediated by interaction with the cognate uncharged tRNA . It i...