The structure and function of the HIV-1 frameshift site stem-loop

Human immunodeficiency virus type I (HIV-I) requires a programmed -1 ribosomal frameshift for translation of nearly one-third of its genome. This frameshifting event is driven by two elements in the HIV-1 messenger RNA: a heptanucleotide slippery sequence (UUUUUUA) followed by a downstream 11 base-pair stem-loop. Together, these elements induce frameshifting during approximately 5% of translation events of the structural open reading frame. This frequency ultimately dictates the ratio of structural (0 frame) to enzymatic (-1 frame) proteins produced. The goal of my work is to determine the relationship between the stem-loop structure and its function in translational reprogramming.;First, I studied the functional role of the 11 base-pair stem-loop during the -1 frameshift event. A strong correlation between frameshift efficiency and the local thermodynamic stability of the first 3 - 4 base-pairs in the stem-loop base was observed. My data demonstrated that frameshifting is driven by a "thermodynamic block" to translocation: the stronger the thermodynamic block, the greater the amount of frameshifting. This thermodynamic block is determined by the local stability of stem-loop base-pairs proximal to the ribosomal mRNA entrance tunnel during frameshifting.;In HIV-1, the frameshift site stem-loop folds into an extended structure, with the 11 base-pair stem-loop separated by a three-purine bulge from an 8 base-pair helix. I used this RNA as a model to investigate the dynamic properties of RNAs with purine-rich bulges, a common structural motif. Using NMR spectroscopy, the interhelical dynamics within this extended structure were examined as a function of potassium and magnesium concentration. The stem-loop was found to undergo large-scale interhelical motions under low-ionic strength conditions that are quenched by physiological concentrations of magnesium. Comparison of my results to previous studies suggested that the impact of monovalent and divalent ions on RNA domain dynamics is a general phenomenon and independent of bulge sequence.