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1H-NMR characterization of L-tryptophan binding to TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis.

ABSTRACT: A 1H-NMR study of the binding of L-tryptophan to the trp RNA-binding attenuation protein of Bacillus subtilis (TRAP), an ondecamer (91.6 kDa), has been implemented. The assignment of the aromatic indole ring proton resonances of the bound tryptophan ligand has been successfully carried out by two-dimensional chemical exchange experiments. The observation of only a single set of chemical shifts of the bound ligand demonstrates that the tryptophan binding site is identical in all the 11 subunits of the protein. Further, the large change in ligand chemical shifts suggests that the conformation of tryptophan ligand undergoes a significant rearrangement after complex formation with TRAP. This is further substantiated by the extensive ligand-induced chemical shift changes observed to the protein resonances and identification of several strong ligand-protein intermolecular nuclear Overhauser effects. A correlation of these preliminary NMR data with the X-ray crystal structure of the TRAP-tryptophan complex also suggests, tentatively, that the observed changes to the NMR spectra of the protein might correspond to changes associated with residues surrounding the tryptophan binding pocket owing to complex formation.

SUBMITTER: Ramesh V

PROVIDER: S-EPMC1217291 | biostudies-other | 1996 May

REPOSITORIES: biostudies-other

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Project description:Transcription of the tryptophan (trp) operon in Bacillus subtilis is regulated by an attenuation mechanism. Attenuation is controlled by the trpRNA-binding attenuation protein (TRAP). TRAP binds to a site in the 5' leader region of the nascent trp transcript in response to the presence of excess intracellular tryptophan. This binding induces transcription termination upstream of the structural genes of the operon. In prior attenuation models, the role of TRAP was only to alter the secondary structure of the leader region RNA so as to promote formation of the trp attenuator, which was presumed to function as an intrinsic terminator. However, formation of the attenuator alone has been shown to be insufficient to induce efficient termination, indicating that TRAP plays an additional role in this process. To further examine the function of TRAP, we performed a genetic selection for mutant TRAPs that bind tryptophan and RNA but show diminished termination at the trp attenuator. Five such TRAP mutants were obtained. Four of these have substitutions at Glu60, three of which are Lys (E60K) substitutions and the fourth of which is a Val (E60V) substitution. The fifth mutant obtained contains a substitution at Ile63, which is on the same β-strand of TRAP as Glu60. Purified E60K TRAP binds tryptophan and RNA with properties similar to those of the wild type but is defective at inducing termination at the trp attenuator in vitroIMPORTANCE Prior models for attenuation control of the B. subtilis trp operon suggested that the only role for TRAP is to bind to the leader region RNA and alter its folding to induce formation of an intrinsic terminator. However, several recent studies suggested that TRAP plays an additional role in the termination mechanism. We hypothesized that this function could involve residues in TRAP other than those required to bind tryptophan and RNA. Here we obtained TRAP mutants with alterations at Glu60 that are deficient at inducing termination in the leader region while maintaining tryptophan and RNA binding properties similar to those of the WT protein. These studies provide additional evidence that TRAP-mediated transcription termination at the trp attenuator is neither intrinsic nor Rho dependent.

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1H-NMR characterization of L-tryptophan binding to TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis.

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