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

Abstract

Anti-TRAP (AT) is a small zinc-binding protein that regulates tryptophan biosynthesis in Bacillus subtilis by binding to tryptophanbound trp RNA-binding attenuation protein (TRAP), thereby preventing it from binding RNA, and allowing transcription and translation of the trpEDCFBA operon. Crystallographic and sedimentation studies have shown that AT can homooligomerize to form a dodecamer, AT 12 , composed of a tetramer of trimers, AT 3 . Structural and biochemical studies suggest that only trimeric AT is active for binding to TRAP. Our chromatographic and spectroscopic data revealed that a large fraction of recombinantly overexpressed AT retains the N-formyl group (fAT), presumably due to incomplete N-formyl-methionine processing by peptide deformylase. Hydrodynamic parameters from NMR relaxation and diffusion measurements showed that fAT is exclusively trimeric (AT 3 ), while (deformylated) AT exhibits slow exchange between both trimeric and dodecameric forms. We examined this equilibrium using NMR spectroscopy and found that oligomerization of active AT 3 to form inactive AT 12 is linked to protonation of the amino terminus. Global analysis of the pH dependence of the trimer-dodecamer equilibrium revealed a near physiological pK a for the N-terminal amine of AT and yielded a pH-dependent oligomerization equilibrium constant. Estimates of excluded volume effects due to molecular crowding suggest the oligomerization equilibrium may be physiologically important. Because deprotonation favors "active" trimeric AT and protonation favors "inactive" dodecameric AT, our findings illuminate a possible mechanism for sensing and responding to changes in cellular pH.allostery | thermodynamic linkage | n-formyl methionine I n bacilli, the trp RNA-binding attenuation protein (TRAP) is responsible for responding to intracellular levels of tryptophan and regulating transcription (1-4) and translation (5-10) of six tryptophan biosynthetic genes clustered in the trpECDFBA operon (1, 11). Transcription of the gene is controlled by an attenuation mechanism, involving competing terminator and antiterminator RNA structural elements in the 5′ leader region of the mRNA. In the absence of cellular tryptophan, the antiterminator structure is favored, allowing transcription and translation of the operon. When cellular tryptophan levels are high, the amino acid binds to TRAP, which becomes activated for binding to an RNA site that overlaps the antiterminator sequence, favoring formation of the terminator; translation of these genes is regulated by a similar mechanism (1,6,9,10,(12)(13)(14). TRAP is a small protein (∼74 amino acids) that assembles into an undecameric (11-mer) ring-shaped structure, with binding sites for tryptophan arranged between neighboring TRAP protomers (15, 16). RNA binds to TRAP by wrapping around the outside of this ring (17).In Bacillus subtilis (Bsu), the protein anti-TRAP (AT), encoded by the yczA gene, provides further regulation by inhibiting mRNA binding by tryptophan-activated TRAP (13,18). AT is a zinc-bind...

Gene regulation by substoichiometric heterocomplex formation of undecameric TRAP and trimeric anti-TRAP

“…AT expression is up-regulated in response to Trp depletion through sensing of elevated levels of uncharged tRNA Trp via a T box antitermination mechanism (6,8). Quantitative Western blotting of cell lysates at conditions mimicking Trp starvation indicate that AT is maximally expressed to fewer than two AT 3 per TRAP 11 and that at these substoichiometric ratios AT is maximally able to restore trpECDFBA operon transcription to 70-80% that of TRAP-free levels (9). Order-of-addition experiments demonstrate that AT inhibition of the TRAP-RNA interaction is determined kinetically, because AT can block RNA binding if present before incubation with RNA but cannot compete effectively with RNA in a preformed complex (7).…”

“…Analytical ultracentrifugation of mixtures of TRAP and AT has resulted in confusing and contradictory interpretations of their interaction (12,13). In one case, sedimentation data suggested that AT and TRAP form large aggregates (12), with the largest occurring at low AT 3 :TRAP 11 ratios, but a second study under alkaline conditions suggested much smaller complexes, consistent with one or more AT 3 s bound to a single TRAP oligomer (13). Interpretation of these results is complicated further by the effect of pH upon the 4AT 3 ↔ (AT 3 ) 4 equilibrium.…”

“…The available structural models of the TRAP-AT complex have remained insufficient to clarify crucial mechanistic matters such as the observation that substoichiometric ratios of AT 3 are sufficient to inhibit the RNA-binding activity of TRAP 11 in vivo (9,15). The affinity of Trp-TRAP for the full-length trp leader RNA (11 G/UAG repeats) has been shown by filter-binding Significance Noncovalent interactions between proteins modulate their functions and occur widely in biological regulation.…”

“…At physiological pH, AT 3 further associates into a tetramer of trimers, (AT 3 ) 4 , via ion pairs involving the protonated N terminus of the protein and conserved aspartate carboxylates (6,11,12). Because it is the trimeric form of AT that is competent for Trp-TRAP binding (13), this equilibrium underlies a means for oligomerization-mediated pH sensing (11). Complications arising from the oligomeric and stoichiometric asymmetry between TRAP 11 and AT 3 were overcome partially by crystallization of TRAP in a dodecameric form, TRAP 12 (13).…”

“…AT comprises an N-terminal zinc-binding domain and a C-terminal helix, which serves as the assembly point for three protomers into a trimer, AT 3 , via a helical bundle (10). At physiological pH, AT 3 further associates into a tetramer of trimers, (AT 3 ) 4 , via ion pairs involving the protonated N terminus of the protein and conserved aspartate carboxylates (6,11,12). Because it is the trimeric form of AT that is competent for Trp-TRAP binding (13), this equilibrium underlies a means for oligomerization-mediated pH sensing (11).…”