BINDING OF HOECHST-33258 TO THE TAR RNA OF HIV-1 - RECOGNITION OF A PYRIMIDINE BULGE-DEPENDENT STRUCTURE

The transactivation response region (TAR) RNA is an essential componen t in transcriptional regulation of the human immunodeficiency virus ty pe-1 (HIV-1) genome. We have examined the interaction between TAR RNA and the bisbenzimidazole derivative Hoechst 33258. Previous studies ha ve shown that this drug, which is well known as an AT-Selective DNA mi nor groove binder, can also interact with CC-rich sequences in DNA as well as with RNA, possibly by intercalation. Absorption spectroscopy, circular dichroism and electric linear dichroism, as well as RNase A f ootprinting, were employed to compare binding of Hoechst 33258 to wild -type RNA and its analogue lacking the pyrimidine bulge, The uridine b ulge, which is an important contributor to the structural stability of TAR, plays an essential role In drug binding. Deletion of the bulge d estabilizes both free and drug-bound forms of TAR and markedly affects the orientation-of the drug chromophore complexed with the RNA. Accor ding to the linear dichroism data, the bisbenzimidazole is oriented mo re or less perpendicular to the RNA helix axis. The data are compatibl e with a model in which the bisbenzimidazole chromophore is inserted i nto the existing cavity created by the pyrimidine bulge. The footprint ing experiments, showing that the drug binds to a unique site opposite the unpaired uridine residues, also support this model. The binding o f Hoechst 33258 to the sequence 5'-GCUCU, which delimits the cavity, r eflects the greater accessibility of that region compared with other s ites in the RNA molecule. The identification of a binding site for sma ll molecules in TAR offers promising perspectives for developing drugs that would block the access of TAR RNA to proteins and therefore for the design of anti-HIV agents.