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

Angela Valbuzzi,Paul Gollnick,Paul Babitzke,Charles Yanofsky

doi:10.1074/jbc.m111813200

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 degrees C demonstrated that AT is active at this elevated temperature.

Highlights

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]
Cross-linking experiments performed previously with the wild type anti-TRAP protein (AT) and TRAP proteins of B. subtilis suggested that AT recognizes a structural feature of TRAP that is formed when this protein binds tryptophan [24]
We extended this finding by examining a group of well characterized B. subtilis TRAP mutant proteins that are altered in tryptophan binding and/or RNA binding [19, 20]

Summary

EXPERIMENTAL PROCEDURES

Protein Purification—The AT protein was purified following a modification of the original procedure [24]. After centrifugation to remove insoluble proteins, the supernatant was dialyzed versus buffer B (50 mM Tris1⁄7HCl, pH 7.8, 1 mM dithiothreitol) overnight. Wild type and mutant TRAP proteins were purified as described previously [19, 20]. Transcription reaction mixtures (10 ␮l) contained B. subtilis vegetative (␴A) RNA polymerase, 10 nM DNA template, 340 nM TRAP protein (wild type, mutant, or from different bacilli species, depending on the experiment), 0.5 mM L-tryptophan, 20 units of RNasin (Promega), and ribonucleoside triphosphates (2.7 mM ATP, 0.7 mM CTP, 1.1 mM GTP, 1.4 mM UTP, 10 ␮Ci ϭ 370 Bq of [␣-33P]UTP 3000 Ci/mmol). Reaction mixtures (20-␮l final volume) contained 412 nM wild type or mutant TRAP and 2.1 ␮M AT, in the presence or absence of 0.5 mM L-tryptophan, in 20 mM Tris-HCl, pH 8, 20 mM NaCl, 4 mM MgCl2. Reactions were terminated by adding SDS sample buffer and boiling for 1 min. Modifications of the standard assay are described in the figure legend

RESULTS

None None None

Wild type

DISCUSSION