On the use of phasing experiments to measure helical repeat and bulge loop-associated twist in RNA.
In a phasing experiment, two bends are introduced into a long duplex RNA or DNA and the number of base pairs between them varied. When electrophoresed in a gel, the set of molecules may show a periodic variation in mobility that contains information about the twist associated with the bends and the intervening helix. We show how a set of three phasing experiments can be used to extract this information, and apply it to an RNA helix bend at the bulge sequence A2. The bulge introduces a negative (left-handed) twist of approximately 30 degrees; at low temperatures, it is mostly confined to the 5' side of the bulge. The apparent helical repeat of random sequence RNA measured in these experiments was 10.2 +/- 0.1 base pairs, an unexpectedly low value. It is likely that moderate curvative of the RNA helix axis (30-40 degrees over 80 bp) has affected the measurement.
Tang
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Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218.
Draper
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D001482:Base Composition; D004247:DNA / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D009690:Nucleic Acid Conformation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012313:RNA / Q000737:chemistry*; D012091:Repetitive Sequences, Nucleic Acid; D013696:Temperature
D012313:RNA; D004247:DNA