Sequence Effects on RNA Bulge-Induced Helix Bending and a Conserved Five-Nucleotide Bulge from the Group I Introns

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Abstract
Bulge loops introduce bends in RNA double helices. Thus, a role for bulge loops in the tertiary folding of RNA is to orient helical elements. The location, size, and sequence of a five-nucleotide bulge are conserved in many of the self-splicing group I introns. We have used gel electrophoretic analysis of helix bending to test the hypothesis that this bulge loop is conserved to control the angle between the flanking helices. Interruption of an RNA duplex by the five-nucleotide bulge of the group I intron from Tetrahymena thermophila results in an electrophoretically retarded species, indicative of bending by the bulge. However, mutation of conserved bases in the bulge has a small effect on the retardation, suggesting that the average induced bend angle is not strongly dependent on the conserved sequence. Electrophoretic analysis of a mixture of bulged duplexes containing all five-nucleotide bulges reveals that most five-nucleotide bulge sequences induce bends that are similar to the bend induced by the conserved bulge. We have calibrated relative electrophoretic mobilities with bends of known magnitude, and characterized the distribution of bulge sequences among bend angles. Though the entire range of bend angles induced by different five-nucleotide bulges is from approximately 45° to 75°, most (>85%) five-nucleotide bulge loops induce bends between 65° and 75°. We have identified several of the anomalous five-nucleotide bulge sequences that induce bends of magnitude smaller than 65°. They are generally, though not universally, pyrimidine-rich.