The crystal structure of the trp RNA-binding attenuation protein of Bacclius subtilis solved at 1.8 A resolution reveals a novel structural arrangement in which the eleven subunits are stabilized through eleven intersubunit beta-sheets to form a beta-wheel with a large central hole. The nature of the binding of L-tryptophan in clefts between adjacent beta-sheets in the beta-wheel suggests that this binding induces conformational changes in the flexible residues 25-33 and 49-52. It is argued that upon binding, the messenger RNA target forms a matching circle in which eleven U/GAG repeats are bound to the surface of the protein ondecamer modified by the binding of L-tryptophan.

MtrB regulates transcription attenuation of the Bacillus subtilis typ operon. We have shown that MtrB, either from B. subtdis or overexpressed in Escherichwa cohl, binds specifically to RNA from the leader region of the tqp operon by a gel mobility-shift assay. This binding is tryptophan dependent. MtrB binds to a transcript terminated at the trp attenuator (-2 to +138) or a read-through transcript (-2 to +318). MtrB does not bind antisense tbp leader RNA or single-stranded trp leader DNA. These results support the model in which attenuation is controlled by tryptophanactivated MtrB influencing the secondary structure of the leader region transcript to form a terminator structure.An unusual form of attenuation is believed to be the sole means of regulating transcription of the Bacillus subtilis tryptophan (trpEDCFBA) operon in response to L-tryptophan (1). Two alternative RNA secondary structures, which form in the 5' leader region RNA transcript, control transcription of the operon (1, 2). In the presence of tryptophan, a terminator structure forms, halting transcription in the leader region, whereas in the absence of tryptophan, an antiterminator structure forms, allowing transcription of the operon. The unusual aspect of this attenuation mechanism is that selection between the alternative RNA structures is mediated by a tryptophan-activated trans-acting regulatory factor rather than by synthesis of a leader peptide, such as occurs in the Escherichia coli trp operon. This regulator appears to function by binding to a specific target in the antiterminator RNA stem and, when bound, to promote formation of the transcription terminator structure (1, 2). The product of the methyltryptophan-resistance (mtr) locus is thought to be the regulatory factor because mutations in this locus eliminate control of the trp operon, resulting in constitutive production of the tryptophan biosynthetic enzymes (1-3). The mtr locus from B. subtilis has been cloned and sequenced and is a two-gene operon consisting of mtrA and mtrB (4). Recently, MtrA (22 kDa) has been identified as GTP cyclohydrolase I, and MtrB (8 kDa) has been shown to be the regulator of the trp operon (5).Evidence that the regulator interacts with an RNA target first came from in vivo studies in which overexpressing the trp leader RNA transcript in trans resulted in constitutive expression of the trp operon, presumably by titrating the regulatory MtrB factor (1, 2). Multiple copies of the trp leader as DNA did not affect expression of the trp operon, which indicated that the regulator target is RNA. Deletion analysis of the leader region localized the regulator target to a 10-base sequence (AGAAUGAGUU), which is repeated twice in the leader transcript, including once in the antiterminator stem (2). Deleting either of these sites lessened effectiveness of the region as a target and deleting both repeats completely abolished target function.The current model for attenuation control of the B. subtilis trp operon suggests that tryptophan-activated MtrB binds t...

In Bacillus subtilis, expression of the tryptophan biosynthetic genes is regulated in response to tryptophan by an RNA-binding protein called TRAP (trp RNA-binding attenuation protein). TRAP has been shown to contain 11 identical subunits arranged in a symmetrical ring. Kinetic and thermodynamic parameters of the interaction between tryptophan-activated TRAP and trp leader RNA were studied. Results from glycerol gradients and mobility shift gels indicate that two TRAP 11-mers bind to each trp leader RNA. A filter binding assay was used to determine an apparent binding constant of 8.0 ؎ 1.3 ؋ 10 9 M ؊1 (K d ‫؍‬ 0.12 ؎ 0.02 nM) for TRAP and an RNA containing residues ؉36 to ؉92 of the trp leader RNA in 1 mM L-tryptophan at 37°C. The temperature dependence of K app was somewhat unexpected demonstrating that the ⌬H of the interaction is highly unfavorable at ؉15.9 kcal mol ؊1. Therefore, the interaction is completely driven by a ⌬S of ؉97 cal mol ؊1 K ؊1 . The interaction between tryptophan-activated TRAP and trp leader RNA displayed broad salt and pH activity profiles. Finally, the rate of RNA dissociation from the RNA⅐TRAP⅐tryptophan ternary complex was found to be very slow in high concentrations of tryptophan (>40 M) but increased in lower tryptophan concentrations. This suggests that dissociation of tryptophan from the ternary complex is the rate-limiting step in RNA dissociation.In Bacillus subtilis, the genes for tryptophan biosynthesis are regulated in response to tryptophan by an RNA-binding protein called TRAP (trp RNA-binding attenuation protein; Refs. 1-3). TRAP regulates expression of the trp genes in three ways, all of which involve TRAP binding RNA in a tryptophandependent manner (3). The trp operon (trpEDCFBA), which contains 6 of the 7 genes required for L-tryptophan biosynthesis, is regulated by transcription attenuation within a 204-nucleotide leader region preceding the first structural gene, trpE (1-2). In the presence of L-tryptophan, TRAP binds the nascent trp leader transcript (4) at a series of 11 G/UAG repeats between residues ϩ36 and ϩ91 ( Fig. 1; Refs. 5-7). This binding prevents formation of an anti-terminator secondary structure which allows a transcription terminator to form and transcription halts in the leader region (1, 8). When L-tryptophan is limiting, TRAP does not bind, the anti-terminator forms, and the operon is expressed.TRAP also regulates translation of two trp genes, trpE (2, 9) and trpG (10), apparently by two different mechanisms. Control of trpE translation is believed to be mediated by TRAP binding to the same series of G/UAG repeats in the leader segment of trp mRNAs that have escaped termination at the attenuator. This binding is thought to alter the RNA secondary structure so as to sequester the trpE ribosome binding site in a stem-loop structure, thus reducing translation initiation (2).trpG is the only tryptophan biosynthetic gene not located within the trp operon and is located within a folic acid biosynthetic operon (11). Regulation of trpG translation occurs wh...

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