The many paths to frameshifting: kinetic modelling and analysis of the effects of different elongation steps on programmed –1 ribosomal frameshifting

Liao, Pei-Yu; Choi, Yong Seok; Dinman, Jonathan D.; Lee, Kelvin H.

Protein sequencing was originally employed to generate the simultaneous slippage model, and to confirm that the À1 PRF site for HIV-1 is U UUU UUA located within the gag/ pol overlap (where the P-site of the ribosome during frameshifting is underlined) (1). Interestingly, $70% of the frameshift products contained Phe-Leu (derived from decoding the 0-frame UUU UUA sequence), while $30% of the products contained Phe-Phe (derived from decoding the À1 frame UUU UUU sequence) at the frameshift site (1, 25) . Previous studies suggested that the product with Phe-Phe at the frameshift site could result from slippage of the P-site tRNA alone (1, 25, 26) , i.e. the product predicted by Pathway I, and that the À1 frame aa-tRNA is subsequently recruited to the ribosome. However, the precise mechanism driving this process remained unclear, and no model has been proposed to date explaining the simultaneous formation of different frameshift proteins. Here, we have developed a kinetic model of À1 PRF to explain all of the experimental observations. This model reveals the major steps in the translation elongation cycle that affect À1 PRF, and reconciles all three models of À1 PRF. In addition, À1 PRF efficiency was monitored in vivo using a dual fluorescence reporter (27) and the compositions of different frameshift proteins were analysed by mass spectrometry. The experimental approach was also applied to study Human T-cell leukemia virus type 1 (HTLV) pro-pol frameshift sequence. This is the first study to demonstrate and quantify the ratio of frameshift products incorporating À1 frame A-site tRNA at this À1 PRF sequence. In agreement with the model predictions, experimental perturbation of different translation steps resulted in different levels of À1 PRF efficiency as well as in the relative ratios of two types of frameshift proteins. Our findings demonstrate that all three kinetic pathways are operative during À1 PRF.