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Transcription of the trp operon of Bacillus subtilis and some related species is regulated by transcription attenuation. A tryptophan-activated RNA-binding regulatory protein, TRAP, binds to the leader segment of the trp operon transcript and promotes formation of an RNA terminator structure, causing transcription termination before RNA polymerase can reach the first structural gene. TRAP recognizes a series of (G/U)AG repeats in the antiterminator segment of the trp leader transcript; the antiterminator precedes and partially overlaps the terminator. TRAP binding to the antiterminator prevents its formation, thereby favoring terminator formation (3, 4, 12, 15). The structures of TRAP and the TRAP-RNA complex have been described previously (1, 2). These structures define how the TRAP protein wraps the leader transcript around its surface and prevents the formation of the antiterminator. TRAP also binds to a (G/U)AG repeat region overlapping the Shine-Dalgarno sequence of the trpG segment of the folate transcript; TRAP binding regulates translation of trpG (4, 6, 17).

In an effort to identify all the potential TRAP binding sites in the B. subtilis genome, we designed an appropriate computer program and searched for (G/U)AG repeats (14). Four (G/U)AG repeat regions were identified, two in addition to the (G/U)AG repeats mentioned above, in the trp operon and trpG. One of these TRAP binding sites overlaps the Shine-Dalgarno sequence of a gene of unknown function, ycbK, and presumably is used to regulate translation of the ycbK coding region (14). The second (G/U)AG repeat region overlaps the yhaG Shine-Dalgarno sequence and presumably regulates initiation of synthesis of its encoded polypeptide. The experiments described in this paper were performed to determine whether yhaG expression was regulated by the TRAP protein and to analyze the function of its encoded polypeptide.

Features of the yhaG gene. To determine the location of the promoter responsible for yhaG expression, primer extension analysis was performed to identify the start point of transcription (data not shown). A site was identified which is preceded by sequences which exhibit high similarity to the consensus sequence of the 10 and 35 regions of vegetative B. subtilis promoters (Fig. 1A). There is a short leader sequence of 82 bases upstream of an open reading frame encoding a predicted product containing 172 amino acid residues. The open reading frame is preceded by a sequence closely resembling a Shine-Dalgarno sequence. A sequence encoding a putative transcription terminator is present downstream of the yhaG coding region.

View larger version (42K): [in this window] [in a new window]   FIG. 1.   (A) Features of the yhaG region of the B. subtilis chromosome. Promoter regions and translation start and stop codons are underlined. The start point of transcription is marked by an asterisk and an arrow. The Shine-Dalgarno sequence (SD) is indicated by a dashed underline. A sequence corresponding to a Rho-independent transcription terminator is indicated by a double underline. Trinucleotide repeats constituting a putative TRAP binding site are shaded. Nucleotide coordinates shown are from 1074054 to 1074773 according to the B. subtilis genome database (8). (B) Predicted topological model of the YhaG protein, generated by the TopPred II computer program using a window size of 21 (5). Oval structures represent alpha-helical transmembrane spans. Predicted amino acid residues at the membrane interface are indicated.

Regulation of yhaG expression. To determine whether expression of yhaG is regulated by TRAP in the presence of tryptophan, strains that contained a yhaG'-'lacZ translational fusion were constructed. A 352-bp fragment encompassing the yhaG promoter, leader region, putative TRAP binding site, and the beginning of the yhaG coding region was cloned into the integration vector ptrpBG1-PLK (10, 16), to produce plasmid pJPS648. The fusion was then integrated into the amyE locus of the chromosome. Strains containing this integrated fusion, in different genetic backgrounds that influence TRAP production, were grown in the presence and absence of tryptophan and assayed for -galactosidase activity (11). In an mtrB⁺ strain, yhaG'-'lacZ expression was significantly reduced by the presence of tryptophan in the growth medium (Table 1; rows 1 and 2). Deleting mtrB, the structural gene for TRAP, resulted in elevated expression of the yhaG'-'lacZ translational fusion, regardless of the presence of added tryptophan. These findings establish that yhaG expression is regulated in response to tryptophan and that the TRAP protein is required for this regulation. Considering the location of the TRAP binding site in the yhaG transcript (Fig. 1A), it is likely that yhaG is translationally regulated by tryptophan-activated TRAP.

The effect of overexpression of yhaG on trp operon and trpG expression. If the yhaG transcript contains a TRAP binding site, then overproduction of this transcript should result in increased expression of the trp operon and of trpG of the folate operon. It was shown previously that overexpression of the TRAP binding site of the trp operon leader region results in increased expression of the trp operon (9). The same 352-bp fragment used to construct the yhaG'-'lacZ fusion was cloned into the multicopy shuttle vector pHY300PLK (7). The resulting plasmid, pJPS647, was introduced into strains which contained integrated trpE'-'lacZ and trpG'-'lacZ reporters. It is evident that both trpE'-'lacZ expression and trpG'-'lacZ expression were increased appreciably by the presence of pJPS647 (Table 1, rows 3 to 10). Addition of tryptophan had no effect on expression of either reporter. The presence of the parental plasmid pHY300PLK alone had no effect. These findings demonstrate that the presence of a multicopy plasmid containing a fragment from yhaG results in considerable relief of TRAP regulation. This presumably occurs because the yhaG transcripts that are produced from the plasmid bind to and remove some of the available TRAP.

Analysis of the likely function of the YhaG protein. On the basis of hydropathy profiles, it appears that the product of the yhaG gene is a polytopic protein with intervening hydrophobic and hydrophilic regions. We suggest that the YhaG protein may be a membrane-embedded protein containing six or possibly seven transmembrane spans. One possible topological model predicted by the TopPred II computer program (5) is presented in Fig. 1B. We therefore considered the possibility that YhaG is a tryptophan transport protein or an aromatic amino acid transporter. The YhaG protein is not homologous to known aromatic amino acid transporters of Escherichia coli or other organisms.

Conclusions. A gene of B. subtilis of previously unknown function specifies a transcript that has a binding site for tryptophan-activated TRAP. Overproduction of the yhaG transcript increases expression of the trp operon and trpG, indicating that this transcript can sequester TRAP in vivo. We have assigned a stretch of nine (G/U/A)AG trinucleotide repeats as a putative TRAP binding site, but further experiments are necessary to ascertain the exact contacts made between TRAP and the yhaG transcript. The predicted TRAP binding site in the yhaG transcript overlaps the Shine-Dalgarno sequence of the yhaG coding region. TRAP was shown to regulate yhaG expression translationally, presumably by binding at this Shine-Dalgarno sequence. TRAP does not regulate transcription of yhaG. The predicted structure of the YhaG protein is that of a transmembrane protein, raising the possibility that YhaG is a transport protein. Amino acid analog resistance-sensitivity tests and labeled tryptophan uptake analyses suggest that the YhaG protein transports tryptophan. Additional experiments are required to unambiguously confirm the function of this protein.