RNA



METHODS
METHODS

Lead(II) as a probe for investigating RNA structure in vivo


MAGNUS  LINDELL  a1, PASCALE  ROMBY  a2 and E. GERHART H.  WAGNER  a1 c1
a1 Institute of Cell and Molecular Biology, Biomedical Center, Uppsala University, 751 24 Uppsala, Sweden
a2 Unité Propre de Recherche 9002 du Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire et Cellulaire, F-67084 Strasbourg Cedex, France

Abstract

In this communication, we describe a simple and reliable method for RNA structure determination in vivo, using the divalent ion, lead(II), as a structural probe. Lead(II) is known to cleave RNA within single-stranded regions, loops, and bulges, whereas cleavages in double-stranded regions are weaker or absent. Because the ion easily entered bacterial cells, Escherichia coli cultures were treated by addition of 50–100 mM lead(II) acetate for 3–7 min, resulting in partial cleavage of RNA in vivo. Cleavage positions were mapped by reverse transcription analysis of total extracted RNA. Three RNAs were analyzed: tmRNA, CopT (the target of the antisense RNA CopA), and the leader region of the ompF mRNA. All three RNAs had previously been analyzed in vitro, and secondary structure models were available. The results presented here show that lead(II) cleavages in vivo yield detailed structural information for these RNAs, which was in good agreement with the models proposed based on in vitro work. These data illustrate the potential of lead(II) as a sequence-independent RNA structure probe for use in living cells.

(Received December 12 2001)
(Revised January 14 2002)
(Accepted February 4 2002)


Key Words: antisense RNA; in vivo mapping; lead(II) acetate; RNA structure; tmRNA.

Correspondence:
c1 Reprint requests to: E. Gerhart H. Wagner, Institute of Cell and Molecular Biology, Uppsala University, Husargatan 3, S-75124, Sweden; e-mail: [email protected].