The Anti-trp RNA-binding Attenuation Protein (Anti-TRAP), AT, Recognizes the Tryptophan-activated RNA Binding Domain of the TRAP Regulatory Protein

Abstract

In Bacillus subtilis, the trp RNA-binding attenuation protein (TRAP) regulates expression of genes involved in tryptophan metabolism in response to the accumulation of L-tryptophan. Tryptophan-activated TRAP negatively regulates expression by binding to specific mRNA sequences and either promoting transcription termination or blocking translation initiation. Conversely, the accumulation of uncharged tRNA Trp induces synthesis of an anti-TRAP protein (AT), which forms a complex with TRAP and inhibits its activity. In this report, we investigate the structural features of TRAP required for AT recognition. A collection of TRAP mutant proteins was examined that were known to be partially or completely defective in tryptophan binding and/or RNA binding. Analyses of AT interactions with these proteins were performed using in vitro transcription termination assays and cross-linking experiments. We observed that TRAP mutant proteins that had lost the ability to bind RNA were no longer recognized by AT. Our findings suggest that AT acts by competing with messenger RNA for the RNA binding domain of TRAP. B. subtilis AT was also shown to interact with TRAP proteins from Bacillus halodurans and Bacillus stearothermophilus, implying that the structural elements required for AT recognition are conserved in the TRAP proteins of these species. Analyses of AT interaction with B. stearothermophilus TRAP at 60°C demonstrated that AT is active at this elevated temperature.The trp RNA-binding attenuation protein (TRAP) 1 of Bacillus subtilis coordinately regulates expression of the genes of tryptophan metabolism in response to the intracellular level of free L-tryptophan (1). Six of the seven trp biosynthetic genes are clustered in the trpECDFBA operon, whereas the seventh, trpG, is located in the folate operon (2). TRAP also regulates expression of yhaG (3), a gene specifying a protein presumably involved in tryptophan transport, and possibly another gene, of unknown function, ycbK (4). The mechanisms by which tryptophan-activated TRAP regulates expression of all of these target genes depend on its ability to bind a specific RNA sequence, designated a TRAP binding site. The site is composed of multiple (G/U)AG repeats, generally separated by 2 nucleotides (5). By binding to this site in trp leader RNA, tryptophan-activated TRAP promotes formation of a terminator structure, as opposed to the alternative antiterminator structure (6). This causes transcription termination in the leader region upstream of the trp operon structural genes (7). TRAP binding has a second effect on trp operon expression; when bound to the leader segment of the trp operon read-through transcript, it stabilizes an RNA hairpin structure that sequesters the trpE Shine-Dalgarno sequence, reducing trpE translation (8, 9). This may influence expression of the entire operon, via translational coupling and transcriptional polarity (10). Regulation of trpG, and presumably yhaG and ycbK, also occurs at the translational level. In the translation initiation segments of...

Translation Control of trpG from Transcripts Originating from the Folate Operon Promoter of Bacillus subtilis Is Influenced by Translation-Mediated Displacement of Bound TRAP, While Translation Control of Transcripts Originating from a Newly Identified trpG Promoter Is Not

“…TRAP regulates the expression of tryptophan metabolism genes by transcription attenuation (trpEDCFBA operon) and translation control (trpG, trpP, ycbK, and trpE) mechanisms. rtpA encodes anti-TRAP (AT), a protein that binds to and inactivates tryptophan-activated TRAP (25). PLK.…”

Crystal structure of Bacillus subtilis anti-TRAP protein, an antagonist of TRAP/RNA interaction

“…Expression of the at operon is controlled by tandem transcription and translation regulatory mechanisms based on sensing the level of charged͞uncharged tRNA Trp (17). Transcription of the structural genes of the at operon is regulated via the T-box transcription antitermination mechanism, where elevated levels of uncharged tRNA Trp induce transcription of rtpA (18), leading to AT production and increased trp operon expression and tryptophan biosynthesis (19). Translational control of at operon expression is provided by the ability of a translating ribosome to move along the mRNA region encoding a tryptophan-rich leader peptide.…”

“…In TRAP͞RNA complexes, the 11 (G͞U)AG repeats of the RNA chain bind to 11 binding sites on the surface of the protein and form a belt around the protein ring (23)(24)(25). A study with mutant TRAP proteins revealed that AT forms a complex only with tryptophan-activated TRAP and appears to recognize a region that is also involved in RNA binding (19). These observations led to the proposal that AT prevents the TRAP͞RNA interaction by masking RNA-binding sites of TRAP (19).…”

“…A study with mutant TRAP proteins revealed that AT forms a complex only with tryptophan-activated TRAP and appears to recognize a region that is also involved in RNA binding (19). These observations led to the proposal that AT prevents the TRAP͞RNA interaction by masking RNA-binding sites of TRAP (19). Interestingly, after TRAP has bound to RNA, AT is unable to disrupt the TRAP͞RNA complex (19).…”

“…These observations led to the proposal that AT prevents the TRAP͞RNA interaction by masking RNA-binding sites of TRAP (19). Interestingly, after TRAP has bound to RNA, AT is unable to disrupt the TRAP͞RNA complex (19). In analytical ultracentrifugation experiments, a complex containing 12 AT subunits per each TRAP 11-mer was observed (26).…”