Determination of the HTLV-1 pro-pol frameshift site RNA secondary structure

Expression of human t-cell leukemia virus type I (HTLV-I) enzymes requires two -1 programmed ribosomal frameshifts. These events occur between the gag-pro and pro-pol open reading frames. Each frameshift site includes a heptanucleotide slippery sequence followed by a downstream structure, which act in cis to produce specific frameshift efficiencies. While -1 PRF and the slippery sequences of these frameshift sites have been established in HTLV-I, their frameshift efficiencies and the structures within these sites have not been determined. In pro-pol frameshift site, an RNA pseudoknot is predicted to fold downstream of the UUUAAAC slippery sequence. However, no structural data exist for this RNA. Here, we report a preliminary structure of the HTLV-1 pro-pol frameshift site RNA. Nucleotide reactivity data acquired from selective 2′-hydroxyl acylation experiments analyzed by primer extension and thermodynamics-based secondary structure predictions are consistent with a pseudoknot secondary structure. Additionally, non-denaturing PAGE analysis of the wild-type structure and two mutant RNAs confirmed that the fold of the wild-type RNA is significantly different from RNAs of identical length that cannot form a pseudoknot structure. These results establish the existence of a pseudoknot structure in the HTLV-1 pro-pol frameshift site.