BULGE-INDUCED BENDS IN RNA - QUANTIFICATION BY TRANSIENT ELECTRIC BIREFRINGENCE

Bulges represent one of the most common non-helical elements in RNA, often displaying a strong degree of phylogenetic conservation, both in location and sequence, within larger RNA molecules. Thus, knowledge of the conformation and flexibility of RNA bulges is an important prerequisite for understanding the rules governing the formation of tertiary structure within the larger molecules. In the current investigation, the magnitudes of the bends induced in a 148 base-pair duplex RNA molecule by single, centrally located bulges of varying size (n = 1 to 6) and base composition (A(n) and U-n series) have been determined through the use of transient electric birefringence (TEE). The TEE approach is highly sensitive to the changes in the global shape of RNA (or DNA) helices that accompany the introduction of points of bending or flexibility near the center of the helix. In the current instance, bulge angles deduced from TEE measurements ranged from similar to 7 degrees to similar to 93 degrees, with the angle increasing with increasing n for both A(n) and U-n series. For both A(n) and U-n series in the absence of Mg2+, the angle increment per added nucleotide varied from similar to 20 degrees to similar to 8 degrees as n increased from 1 to 6. These angle increments remained unchanged for the A(n) series in the presence of Mg2+; however, the angle increments for the U-n series were reduced by a factor of 2 for all values of n. Thus the current observations have identified structural transitions in one of the simplest non-helical elements in RNA, transitions that are dependent on both sequence and counterion valence. Finally, the measured bend angles are strongly correlated with the degree of reduction in electrophoretic mobility of bulge-containing RNA helices. The observed correlation was used to obtain a semi-empirical relationship between bend angle and mobility in order that additional angles might be assigned, by interpolation, through the use of gel data alone.