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Nucleic Acids Research, 1991, Vol. 19, No. 11 2971-2977
© 1991

MOLECULAR BIOLOGY

Use of electrophoretic mobility to determine the secondary structure of a small antisense RNA

Jean-Philippe Jacques+ and Miriam M. Susskind*

Department of Biological Sciences, University of Southern California Los Angeles, CA 90089-1340, USA

*To whom correspondence should be addressed

Received February 21, 1991. Revised April 11, 1991. Accepted April 11, 1991.

Natural antisense RNAs have stem-loop (hairpin) secondary structures that are important for their function. The sar antisense RNA of phage P22 is unusual: the 3' half of the molecule forms an extensive stem-loop, but potential structures for the 5' half are not predicted to be thermodynamically stable. We devised a novel method to determine the secondary structure of sar RNA by examining the electrophoretic mobility on non-denaturing gels of numerous sar mutants. The results show that the wild-type RNA forms a 5' stem-loop that enhances electrophoretic mobility. All mutations that disrupt the stem of this hairpin decrease mobility of the RNA. In contrast, mutations that change the sequence of the stem without disrupting it (e.g. change G·U to A·U) do not affect mobility. Nearly all mutations in single-stranded regions of the structure also have no effect on mobility. Confirmation of the proposed 5' stem-loop was obtained by constructing and analyzing compensatory double mutants. Combinations of mutations that restore a base-pair of the stem also restore mobility. The genetic phenotypes of sar mutants confirm that the proposed secondary structure is correct and is essential for optimal activity of the antisense RNA in vivo.

+Present address: Department of Microbiology, University of Geneva Medical School, 9 Ave de Champel, CH1211 Geneva, Switzerland


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