RESUMO
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 x 10(9) m-1 (Kd = 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 degrees C. The temperature dependence of Kapp was somewhat unexpected demonstrating that the delta H of the interaction is highly unfavorable at + 15.9 kcal mol-1. Therefore, the interaction is completely driven by a delta 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 RNA-TRAP.tryptophan ternary complex was found to be very slow in high concentrations of tryptophan (> 40 microM) but increased in lower tryptophan concentrations. This suggests that dissociation of tryptophan from the ternary complex is the rate-limiting step in RNA dissociation.
Assuntos
Bacillus subtilis/química , Proteínas de Bactérias , RNA Mensageiro/química , Proteínas de Ligação a RNA/química , Fatores de Transcrição/química , Sequência de Bases , Primers do DNA , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Sais , Termodinâmica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Triptofano/químicaRESUMO
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.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a RNA/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Bacillus subtilis/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Óperon , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Triptofano/genética , Triptofano/metabolismoRESUMO
The trp RNA-binding attenuation protein (TRAP) regulates expression of the B. subtilis trp operon by binding to an RNA target composed of 11 G/UAG repeats in the trp leader transcript. TRAP contains 11 identical subunits arranged in a ring. The binding of 11 molecules of L-tryptophan to sites formed between adjacent subunits activates TRAP to bind trp leader RNA. We propose that the trp leader RNA forms a matching circle on the surface of TRAP when it binds. RNA binding is relatively insensitive to changes in ionic strength and pH, and is apparently driven by changes in entropy. Two lysine residues on the surface of TRAP have been shown to be important for RNA binding.
Assuntos
Proteínas de Bactérias/metabolismo , RNA Bacteriano/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação , Genes Bacterianos , Lisina/metabolismo , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Óperon , Conformação Proteica , RNA Bacteriano/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Triptofano/metabolismoRESUMO
The trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis has been crystallized and examined by crystallography using X-ray synchrotron radiation diffraction data. Crystals of TRAP complexed with L-tryptophan belong to space group C2 with a = 156.8 A, b = 114.05 A, c = 105.9 A, beta = 118.2 degrees. Crystals of a potential heavy-atom derivative of TRAP complexed with 5-bromo-L-tryptophan grow in the same space group with similar cell dimensions. X-ray data for the native crystals and for the derivative have been collected to 2.9 A and 2.2 A resolution, respectively. Peaks in the self-rotation function and in the Patterson synthesis could only be explained by two 11-subunit oligomers (each formed by an 11-fold axis of symmetry) in the asymmetric unit lying with the 11-fold rotation axes parallel to each other. The consequence is that the TRAP molecule has 11-fold symmetry and contains 11 subunits.
Assuntos
Bacillus subtilis/química , Proteínas de Bactérias/química , Proteínas de Ligação a RNA/química , Fatores de Transcrição/química , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Ciclotrons , Modelos Químicos , Óperon/genética , Conformação Proteica , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Triptofano/análogos & derivados , Triptofano/químicaRESUMO
MtrB regulates transcription attenuation of the Bacillus subtilis trp operon. We have shown that MtrB, either from B. subtilis or overexpressed in Escherichia coli, binds specifically to RNA from the leader region of the trp 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 trp leader RNA or single-stranded trp leader DNA. These results support the model in which attenuation is controlled by tryptophan-activated MtrB influencing the secondary structure of the leader region transcript to form a terminator structure.
