The influence of symmetric internal loops on the flexibility of RNA

Abstract

Internal loops are structural elements, often highly conserved, that are found in many RNA molecules of biological importance. They consist of short stretches of sequence in which the bases in one strand are not able to form canonical pairs with bases in the other strand, and are bounded on either side by helical RNA. In an effort to examine the influence of internal loops on the relative angular orientations of the flanking helices, we have quantified the apparent bend angles for symmetric internal loops of the form A(n)-A(n) and U(n)-U(n) (n=2, 4, and 6), located at the center of 150 to 154 bp RNA molecules, using the method of transient electric birefringence. This hydrodynamic method exploits the extreme sensitivity of the rate of rotational reorientation of the RNA molecules to the presence and magnitude of internal bends and/or points of increased flexibility. The birefringence decay behavior of the loop-containing RNA molecules was found to be much less strongly influenced by the presence of symmetric internal loops than by bulges of the same sequence and size. This general observation is mirrored by the electrophoretic behavior of the loop-containing molecules, which are much less strongly retarded on polyacrylamide gels than are corresponding, bulge-containing RNA molecules. The apparent bend angles for the symmetric loops range from approximately 20 degrees to 40 degrees as n is increased 2 to 6 with a marginal shift to smaller angles in the presence of Mg2+. The apparent angles were similar when represented either as fixed bends of the specified angles (static representation), or as points of increased flexibility of specified root-mean-square angle (dynamic representation). For the. For the latter representation, the corresponding angular dispersion would correspond to a loop persistence length of approximately 60 to 150 A, compared to 700 A for duplex RNA and depending slightly on sequence and buffer conditions.