Transcriptional approaches to riboswitch studies

Methods Mol Biol. 2009;540:39-51. doi: 10.1007/978-1-59745-558-9_4.

Abstract

Natural RNA sensors of small molecules (a.k.a. riboswitches) regulate numerous metabolic genes. In bacteria, these RNA elements control transcription termination and translation initiation by changing the folding pathway of nascent RNA upon direct binding of a metabolite. To identify and study riboswitches we used in vitro reconstituted solid-phase transcription elongation/termination system. This approach allows for direct monitoring of ligand binding and riboswitch functioning, establishing the working concentration of a ligand as a function of RNA polymerase speed, and also probing RNA structure of the riboswitch. Using this system we have been able to identify and characterize first several riboswitches including those involved in vitamin biosynthesis and sulfur metabolism. The system can be utilized to facilitate biochemical studies of riboswitches in general, i.e., to simplify analysis of riboswitches that are not necessarily involved in transcriptional control.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Bacillus subtilis / genetics
  • DNA, Bacterial / genetics
  • DNA-Directed RNA Polymerases / metabolism
  • Escherichia coli / genetics
  • Molecular Biology / methods*
  • RNA, Untranslated / metabolism*
  • Regulatory Sequences, Ribonucleic Acid / genetics
  • Templates, Genetic
  • Transcription, Genetic*

Substances

  • DNA, Bacterial
  • RNA, Untranslated
  • Regulatory Sequences, Ribonucleic Acid
  • DNA-Directed RNA Polymerases