The nature of the TRAP–Anti-TRAP complex

Abstract

Tryptophan biosynthesis is subject to exquisite control in species of Bacillus and has become one of the best-studied model systems in gene regulation. The protein TRAP (trp RNA-binding attenuation protein) predominantly forms a ring-shaped 11-mer, which binds cognate RNA in the presence of tryptophan to suppress expression of the trp operon. TRAP is itself regulated by the protein Anti-TRAP, which binds to TRAP and prevents RNA binding. To date, the nature of this interaction has proved elusive. Here, we describe mass spectrometry and analytical centrifugation studies of the complex, and 2 crystal structures of the TRAP-Anti-TRAP complex. These crystal structures, both refined to 3.2-Å resolution, show that Anti-TRAP binds to TRAP as a trimer, sterically blocking RNA binding. Mass spectrometry shows that 11-mer TRAP may bind up to 5 AT trimers, and an artificial 12-mer TRAP may bind 6. Both forms of TRAP make the same interactions with Anti-TRAP. Crystallization of wild-type TRAP with Anti-TRAP selectively pulls the 12-mer TRAP form out of solution, so the crystal structure of wild-type TRAP-Anti-TRAP complex reflects a minor species from a mixed population.protein complex ͉ X-ray crystallography ͉ transcription regulation ͉ analytical ultracentrifugation ͉ mass spectrometry T RAP (trp RNA-binding attenuation protein) plays a central role in the highly intricate regulation of transcription and translation of the trp operon in several species of Bacillus, and this system has provided important insights into several mechanisms of gene regulation (1-5). The protein forms an 11-mer ring that binds 11 molecules of tryptophan (Trp) at symmetryrelated sites (6-8), and the TRAP-Trp-RNA complex has been solved for the Bacillus stearothermophilus protein (9). With tryptophan bound, TRAP can bind trp mRNA and induce transcription termination. In Bacillus subtilis, when levels of charged tRNA Trp fall, the protein Anti-TRAP (AT) is expressed, which blocks RNA binding to Trp-bound TRAP and relieves expression of the trp operon (10, 11). AT forms a stable trimer, 4 copies of which can further associate into a dodecamer form (12, 13). Each 53-residue polypeptide chain carries a zincbinding site made of 4 cysteine residues, and chemical crosslinking studies suggested that the active form of AT may be a hexamer (14). Analytical ultracentrifugation data have been interpreted to indicate that the AT dodecamer is the active (TRAP-binding) form, giving an AT 12 -TRAP 11 complex, although it was noted that the data did not rule out alternative stoichiometry (12). Antson and colleagues (13) further proposed, on the basis of their X-ray structure of AT, a model in which the AT dodecamer may bind up to 4 TRAP 11-mer rings by aligning their 11-fold symmetry axes with its 3-fold axes. In this model, AT would not directly block RNA binding, or contact TRAP residues Lys-37 and Arg-58 that are known to be required for TRAP-RNA (15) and TRAP-AT interaction (10). Instead it penetrates the TRAP ring, and it is suggested that there is indirect ...

Mechanism for pH-dependent gene regulation by amino-terminus-mediated homooligomerization of Bacillus subtilis anti- trp RNA-binding attenuation protein

“…It is also interesting to note the conditions that lead to crystal formation of both the AT 12 (21,38) and the AT 3 -TRAP complex (27) are consistent with the pHdependent oligomerization reported in this paper. Bsu AT 12 was crystallized from a pH 6.5 solution (21) where N-terminal protonation would favor formation of the dodecameric form, whereas the AT 3 -TRAP complex was crystallized at a pH of 8.5 (27) where trimer dominates. The present measurements permit a rough analysis of the oligomeric state of AT in bacterial cells.…”

“…Alanine scanning studies of AT highlighted residues important for TRAP binding and showed that dodecameric AT was unlikely to be able to bind to TRAP (26). In agreement with these findings, crystallographic studies of 12-mer-forming variants of TRAP in complex with AT showed six AT trimers (AT 3 ) arranged along the outside of the TRAP ring, suggesting that the trimer is the species that binds to TRAP (27). However, in vivo studies of the relative concentrations of ATand TRAP seem to show that TRAP activity can be fully inhibited by AT present at a ratio of two AT 3 per TRAP ring (28).…”

“…Because structural and biochemical data indicate that only trimeric AT can bind and inhibit TRAP (21,26,27), a cellular shift to low pH would favor AT 12 formation and result in decreased gene expression. The coincidence of the internal pH of growing B. subtilis cells with our measured pK a for deprotonation of the amino terminus of AT 3 (pH 7.4) indicates that changes in cellular pH could alter the trimer-dodecamer equilibrium and thereby alter gene expression profiles.…”

Positions of Trp Codons in the Leader Peptide-Coding Region of the at Operon Influence Anti-Trap Synthesis and trp Operon Expression in Bacillus licheniformis

“…Depending on how many TRAP molecules per cell are Trp activated and AT free, TRAP will be partially or fully active. Thus, TRAP can bind up to 11 molecules of Trp, to some extent cooperatively (4,25,36,43), and each bound Trp molecule can contribute to TRAP's RNA-binding ability. However, even when TRAP is fully activated, AT can bind to TRAP and block TRAP's RNAbinding ability.…”

“…When free Trp is plentiful and available, it binds to-and activates-TRAP. The TRAP protein contains 11 identical protein subunits, 11 Trp binding sites, and 11 Lys-Lys-Arg motifs on the periphery of the protein complexto Trp-activated TRAP at its RNA-binding surface (35,36,43) and preventing TRAP from binding to its target RNAs. Thus, by binding to TRAP, AT can regulate transcription or translation of all the operons regulated by TRAP (39,40).…”