title: Identification of a Residue (Glu60) in TRAP Required for Inducing Efficient Transcription Termination at thetrpAttenuator Independent of Binding Tryptophan and RNA.
abstract: Transcription of the tryptophan (trp) operon inBacillus subtilisis regulated by an attenuation mechanism. Attenuation is controlled by thetrpRNA-bindingattenuationprotein (TRAP). TRAP binds to a site in the 5' leader region of the nascenttrptranscript 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 thetrpattenuator, 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 thetrpattenuator. 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 thetrpattenuatorin vitroIMPORTANCEPrior models for attenuation control of theB. subtilis trpoperon 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 thetrpattenuator is neither intrinsic nor Rho dependent.
authors.affiliation: Department of Biological Sciences, University at Buffalo, Buffalo, New York, USA.|Department of Microbiology and Immunology, University at Buffalo, Buffalo, New York, USA.
authors.forename: Natalie M
authors.initials: NM
authors.lastname: McAdams
authors.affiliation: Department of Biological Sciences, University at Buffalo, Buffalo, New York, USA.|Molecular Genetics Department, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Andrea
authors.initials: A
authors.lastname: Patterson
authors.affiliation: Department of Biological Sciences, University at Buffalo, Buffalo, New York, USA gollnick@buffalo.edu.
authors.forename: Paul
authors.initials: P
authors.lastname: Gollnick
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C079092:MtrB protein, Bacteria; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D014364:Tryptophan
keywords: RNA binding proteins; termination; transcription; tryptophan operon