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Characterization of TRAP-Mediated Regulation of the B. subtilis trp Operon Using In Vitro Transcription and Transcriptional Reporter Fusions In Vivo

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Book cover RNA Remodeling Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1259))

Abstract

In Bacillus subtilis, transcription of the tryptophan biosynthetic operon is regulated by an attenuation mechanism involving two alternative RNA secondary structures in the 5′ leader region upstream of the structural genes. Regulation is accomplished, at least in part, by controlling which RNA structure forms during transcription of the operon. When intracellular tryptophan levels are high, the trp RNA-binding attenuation protein (TRAP) binds to the nascent trp mRNA to promote formation of a transcription terminator structure so as to induce transcription termination prior to the structural genes. In limiting tryptophan, TRAP does not bind, the alternative antiterminator RNA structure forms, and the operon is transcribed. Several in vitro and in vivo assays have been utilized to study TRAP-mediated regulation of both transcription and translation. Here, we describe using in vitro transcription attenuation assays and in vivo trp-lacZ fusions to examine TRAP-mediated regulation of the trp genes.

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Authors and Affiliations

  1. Department of Biological Sciences, University at Buffalo, 609 Hochstetter Hall, Buffalo, NY, 14260, USA

    Natalie M. McAdams & Paul Gollnick

  2. Department of Microbiology and Immunology, University at Buffalo, Buffalo, NY, 14214, USA

    Natalie M. McAdams

Authors
  1. Natalie M. McAdams
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  2. Paul Gollnick
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Corresponding author

Correspondence to Paul Gollnick .

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Editors and Affiliations

  1. CNRS Centre de Biophysique Moléculaire, Orléans, France

    Marc Boudvillain

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McAdams, N.M., Gollnick, P. (2015). Characterization of TRAP-Mediated Regulation of the B. subtilis trp Operon Using In Vitro Transcription and Transcriptional Reporter Fusions In Vivo. In: Boudvillain, M. (eds) RNA Remodeling Proteins. Methods in Molecular Biology, vol 1259. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2214-7_20

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  • DOI: https://doi.org/10.1007/978-1-4939-2214-7_20

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2213-0

  • Online ISBN: 978-1-4939-2214-7

  • eBook Packages: Springer Protocols

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