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Identification of a Residue (Glu60) in TRAP Required for Inducing Efficient Transcription Termination at the trp Attenuator Independent of Binding Tryptophan and RNA.

Research paper by Natalie M NM McAdams, Andrea A Patterson, Paul P Gollnick

Indexed on: 11 Jan '17Published on: 11 Jan '17Published in: Journal of bacteriology



Abstract

Transcription of the tryptophan (trp) operon in Bacillus subtilis is regulated by an attenuation mechanism. Attenuation is controlled by the trp RNA-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 TRAP proteins 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) and the fourth is Val (E60V). 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 similar properties as wild type but is defective at inducing termination at the trp attenuator in vitro IMPORTANCE: 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 have 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 the WT protein. These studies provide additional evidence that TRAP-mediated transcription termination at the trp attenuator is neither intrinsic nor Rho-dependent.