Solution Structure and Thermodynamic Investigation of the HIV-1 Frameshift Inducing Element

Expression of the HIV reverse transcriptase and other essential viral enzymes requires a −1 translational frameshift. The frameshift event is induced by two highly conserved RNA elements within the HIV-1 mRNA: a UUUUUUA heptamer known as the slippery sequence, and a downstream RNA structure. Here, we report structural and thermodynamic evidence that the HIV-1 frameshift site RNA forms a stem-loop and lower helix separated by a three-purine bulge. We have determined the structure of the 45 nucleotide frameshift site RNA using multidimensional heteronuclear nuclear magnetic resonance (NMR) methods. The upper helix is highly thermostable (Tm>90 °C), forming 11 Watson–Crick base-pairs capped by a stable ACAA tetraloop. The eight base-pair lower helix was found to be only moderately stable (Tm=47 °C). A three-purine bulge separates the highly stable upper helix from the lower helix. Base stacking in the bulge forms a wedge, introducing a 60° bend between the helices. Interestingly, this bend is similar to those seen in a number of frameshift inducing pseudoknots for which structures have been solved. The lower helix must denature to allow the ribosome access to the slippery site, but likely functions as a positioning element that enhances frameshift efficiency.