Analysis of the <i>Bacillus subtilis</i> genome sequence reveals nine new T‐box leaders (1998)

Sir, The T-box family includes some 20 genes of Gram-positive bacteria encoding either aminoacyl-tRNA synthetases or amino acid biosynthesis enzymes. They are preceded by leader regions sharing conserved features (Grundy and Henkin, 1994, J Mol Biol235: 798–804) and are regulated by a common transcription antitermination mechanism involving interaction of the leader transcript with the cognate uncharged tRNA (Grundy et al., 1993, Cell74: 475–482). All T-box leaders 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 sequence, called the T box, is highly conserved. The T-box leader transcripts also have in common other putative stem–loop structures. Most conserved is a complex stem structure, at the 5′ end of the transcript, where a loop, close to the basis of the stem, contains a triplet designated the specifier codon. This codon specifies the amino acid expected to regulate the expression of the system. In addition to the conserved sequences involved in the formation of the terminator/antiterminator structures, several short sequences are conserved, with some degeneracy, in almost all leaders. The conservation of these features suggested a regulatory model in which antitermination is mediated by interaction between the leader transcript and uncharged cognate tRNA. This interaction is likely to involve on one hand, the specifier codon and the anticodon, and on the other, the tRNA acceptor arm and the ‘anti-acceptor’, a 4 nt sequence within the T box as mutations in these regions strongly affect antitermination. Comparison of known T-box sequences revealed that each corresponds to the 5′-AANNNGGGTGGNACCNCG-3′ consensus (where N stands for any nucleotide) with no more than one mismatch. We took advantage of this high sequence conservation to search for other T boxes in the recently published complete genome sequence of B. subtilis (Kunst et al., 1997, Nature390: 249–256). This revealed 20 occurrences of this consensus, among which nine had not been described before (Table 1). Inspection of the sequence flanking the nine newly detected T boxes revealed, in all cases, the presence of the typical features of T-box leaders: (i) the T box is present within a non-coding region, (ii) a typical intrinsic transcription terminator is present closely (13–36 bases) downstream of the T box. The transcript of this region can adopt two mutually exclusive terminator or antiterminator structures, (iii) a start codon is present downstream of the T box, at a distance of 70–148 bases, and (iv) the leader region has the potential to fold into a consensus secondary structure conserved in all T-box leaders. The most conserved features of the structure are the antiterminator stem and the first stem, at the 5′ end of the leader, which contains the specifier codon. Attempts to identify further T boxes in B. subtilis by searching for sequences containing two or three mismatches did not reveal any other typical leader, suggesting that the search was exhaustive. Comparison of both known and newly identified T-box leaders allowed us to establish a detailed structure and sequence consensus (Fig. 1). In addition, to the three stem–loop structures designated I–III by Grundy and Henkin, we identified a fourth conserved one between stems II and III. Previously described sequence motifs CAGAGA and G(G/C)UG(C/A), in single-stranded regions of stem I, were found in newly identified T-box leaders, together with the ‘F-box’ CGUUA (Fig. 1). In addition, we observed two short, conserved motifs: a GA motif at the basis of stem I and a GAG motif in the sequence between stems I and II. . Consensus secondary structure of T-box leaders. This consensus was deduced from analysis of known and newly described (this paper) T-box leaders. A. Leader transcript folded in the antiterminator structure stabilized by interaction with cognate uncharged tRNA. Open circles represent any nucleotide. Length of stems and loops correspond to mean values. Shadowed nucleotides and sequences are highly conserved. The specifier codon, anticodon, anti-acceptor and acceptor arm sequences are boxed. Putative interactions between the tRNA and the leader transcript are indicated by a dotted line. The CGUAA consensus sequence (F box) is found at a variable position, indicated by an arrow, relative to secondary structure. B. 3′ end of the leader transcript folded in the terminator structure. The genes or operons downstream of the nine new T-box leaders were examined (Table 1). Six correspond to known genes, five of which code for aminoacyl-tRNA synthetases (serS, ileS, alaS, hisSaspS, glyQS), and one for the biosynthesis of an amino acid, the proline (proBA). Three correspond to genes without known functions (ycbK, yqjO and yvbW ). Analysis of these last three genes using BLAST revealed that yqjO has 30–45% identity to various bacterial proC genes and most probably represents a B. subtilis proC analogue. yvbW has 28–48% identity to known bacterial ABC transporters involved in the transport of various amino acids, such as the E coli aroP, pheP, lysP and the Salmonella typhimurium proY and therefore probably encodes an amino acid ABC transporter. yczA showed no homology to any known protein in the databases. Genes serS, ileS, alaS and glyQS, encoding seryl-tRNA-, isoleucyl-tRNA-, alanyl-tRNA-synthetase and the two subunits of glycyl-tRNA synthetase, respectively, are preceded by cognate, properly located, specifier codons. It is thus likely that their transcription is regulated by tRNA-mediated antitermination. However, two deviations from the known systems were observed. The AUC (Ile) specifier codon upstream ileS is separated from the downstream base-paired region by two base pairs instead of one. The Ala specifier codon upstream alaS is GCU, which is doubly surprising. First, all previously described B. subtilis specifier codons have a C in their third (‘wobbling’) position, with the exception of the unique UGG (Trp) codon. Second, only tRNAAlaGCA and tRNAAlaGCC exist in B. subtilis (Kunst et al., 1997). This may indicate that antitermination can still occur in the absence of a perfect codon–anticodon interaction, possibly by a phenomenon similar to wobbling. The case of the hisS and aspS genes is interesting as both are separated by 13 nucleotides and are thus likely to be co-transcribed, and the specifier codon upstream is an AAC (Asn) codon. Examination of the leader sequence did not reveal any His or Asp codon properly located. All tRNAHis identified in B. subtilis recognize the CAC codon, and all tRNAAsp recognize the GAC codon. It is therefore possible that either tRNA could participate in the regulation by recognizing the AAC specifier codon. Interestingly, sequence complementarity could be extended by interaction between the A 5′ to the specifier in both leaders, with the U conserved in all tRNAs 5′ to the anticodon. This suggests that transcription of the hisSaspS could be controlled by either tRNAHis or tRNAAsp. Proline biosynthesis in bacteria is carried out by genes proA, proB and proC. In B. subtilis proB and proA are apparently co-transcribed and are preceded by the CCU (Pro) specifier codon, whereas proC is transcribed separately and is preceded by the CCC (Pro) codon. These results suggests that proline biosynthesis is controlled by tRNA-mediated antitermination. Like alaS, the proBA operon is preceded by the CCU specifier codon that has no C in its third position and is not fully complementary to an existing proline anticodon as only tRNAProUGG and tRNAProGGG have been identified in B. subtilis. These observations suggests that some tolerance may take place in the specifier codon/anticodon interaction. Such tolerance could also explain how the hisSaspS operon, encoding for two aminoacyl tRNA synthetases with different specificities, could be controlled by depletion in either His or Asp. Experimental evidence has recently been obtained in a related Lactococcus lactis system, in which perfect fit between the specifier codon and the tRNA anticodon is not essential for antitermination (M. van de Guchte et al.). yvbW encodes a putative ABC amino acid transporter. Such a gene is likely to be induced by limitation in the transported amino acid(s). However, no amino acid transporter has yet been shown to be controlled by a T-box leader. Although a specifier codon stem could easily be recognized upstream this gene, it was not possible to identify a specifier codon unambiguously as the basis of the first stem can form several alternative structures. This feature could render the expression of yvbW responsive to depletion in any of several amino acids. A UGG (Trp) specifier codon could be recognized upstream yczA. As the function of this gene is presently unknown, it is not yet possible to assess the significance of this finding.