Kinking of RNA Helices by Bulged Bases, and the Structure of the Human Immunodeficiency Virus Transactivator Response Element

We have used gel electrophoresis to show that the pyrimidine bulge of the HIV-1 TAR sequence causes a local bending of the helical axis. The TAR bulge caused a retardation in electrophoretic mobility in polyacrylamide gels. When this was placed adjacent to an additional bulged sequence in a linear RNA fragment, the mobility of the molecule varied sinusoidally with the spacing between the two bulges. Electrophoretic mobilities suggested that the TAR sequence context of the pyrimidine bulge causes a greater degree of axial kinking than in an equivalent randomly chosen sequence. Experiments in which an A5 bulge was progressively opposed by adenine bases inserted in the opposite strand showed that even a single opposed adenine markedly reduced electrophoretic mobility, i.e. axial bending, and two adenine bases reduced the mobility virtually to that of a normal duplex. We suggest that the pronounced kinking resulting from an unopposed bulge provides a particularly recognizable feature in RNA, and that this is the basis of the interaction between the HIV Tat protein and the TAR sequence.