Thermodynamic characterization of an engineered tetracycline-binding riboswitch

Riboswitches reflect a novel concept in gene regulation that is particularly suited for technological adaptation. Therefore, we characterized thermodynamically the ligand binding properties of a synthetic, tetracycline (tc)-binding RNA aptamer, which regulates gene expression in a dose-dependent manner when inserted into the untranslated region of an mRNA. In vitro, one molecule of tc is bound by one molecule of partially pre-structured and conformationally homogeneous apo-RNA. The dissociation constant of 770 pM, as determined by fluorimetry, is the lowest reported so far for a small molecule-binding RNA aptamer. Additional calorimetric analysis of RNA point mutants and tc derivatives identifies functional groups crucial for the interaction and including their respective enthalpic and entropic contributions we can propose detailed structural and functional roles for certain groups. The conclusions are consistent with mutational analyses in vivo and support the hypothesis that tc-binding reinforces the structure of the RNA aptamer, preventing the scanning ribosome from melting it efficiently.

• Publication year

  2006

• Venue

  Nucleic Acids Research

• Publication date

  2006-05-17

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1093/nar/gkl347PMID 16707663PMCID 1463898
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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