Luebke K J, Tinoco I
Department of Chemistry, University of California at Berkeley, USA.
Biochemistry. 1996 Sep 10;35(36):11677-84. doi: 10.1021/bi960914r.
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 degrees to 75 degrees, most ( > 85%) five-nucleotide bulge loops induce bends between 65 degrees and 75 degrees. We have identified several of the anomalous five-nucleotide bulge sequences that induce bends of magnitude smaller than 65 degrees. They are generally, though not universally, pyrimidine-rich.
凸起环会在RNA双螺旋结构中引入弯曲。因此，凸起环在RNA三级折叠中的作用是使螺旋元件定向。在许多自我剪接的I组内含子中，一个五核苷酸凸起的位置、大小和序列是保守的。我们利用凝胶电泳分析螺旋弯曲来检验这一假设，即这种凸起环之所以保守是为了控制侧翼螺旋之间的角度。嗜热四膜虫I组内含子的五核苷酸凸起打断RNA双链体后会产生一种电泳迁移率减慢的条带，这表明该凸起会导致弯曲。然而，凸起中保守碱基的突变对迁移率减慢的影响较小，这表明平均诱导弯曲角度并不强烈依赖于保守序列。对包含所有五核苷酸凸起的凸起双链体混合物进行电泳分析发现，大多数五核苷酸凸起序列诱导的弯曲与保守凸起诱导出的弯曲相似。我们已用已知大小的弯曲校准了相对电泳迁移率，并对弯曲角度范围内的凸起序列分布进行了表征。虽然不同的五核苷酸凸起诱导的弯曲角度范围约为45度至75度，但大多数（>85%）五核苷酸凸起环诱导的弯曲角度在65度至75度之间。我们已鉴定出几个异常的五核苷酸凸起序列，它们诱导的弯曲角度小于65度。它们通常富含嘧啶，但并非普遍如此。
