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.

An elegant series of biochemical analyses have established detailed kinetic models of translocation (28) and aa-tRNA selection (29) . Translocation involves EF-G binding to the pre-translocational ribosome, GTP hydrolysis, unlocking conformation change, Pi release, tRNA movement, relocking conformation change and dissociation of EF-G from the post-translocational ribosome. This concept is illustrated along the top of Figure 2 from component PA (pre-translocational ribosome) to E 0 P 0 (post-translocational ribosome). Detailed descriptions for each rate constant are shown in Supplementary Table S1 . Selection and accommodation of aa-tRNA involves initial binding of the ternary complex EF-Tu:aa-tRNA:GTP, codon recognition, EF-Tu GTPase activation, GTP hydrolysis, dissociation of EF-Tu from the ribosome and accommodation of the acceptor end of the aa-tRNA into the A-site or the rejection of the aa-tRNA by proofreading. Detailed descriptions for each rate constant are shown in Supplementary Table S2. In the absence of frameshifting, progression through these steps of the elongation cycle results in synthesis of the non-frameshift protein, called NFS ( Figure 2 ). Our kinetic model suggests three possible reaction pathways that could generate À1 frameshift proteins ( Figure 2 ). In Pathway I, blockage of the mRNA channel entrance by the downstream stimulatory structure induces incomplete translocation with the pre-translocational ribosome positioned at deX XXY. Specifically, the reading frame shift occurs between the tRNA movement and Pi release (rate constant r TL0 ), and the relocking step (rate constant r 45 ). Weiss et al. (23) suggested that when the two tRNAs move from P/E and A/P to the E/E and P/P states, they can un-pair from the mRNA and re-pair with the À1 reading frame. In our model, r t represents the rate constant for a ribosome:EF-G:GDP complex with two tRNAs in the E-and P-sites (E 0 P 0 EFGgdp ) to re-pair with the À1 reading frame (E 02 P 02 EFGgdp ). This motion is reversible, as denoted by the r -t rate constant. This step is followed by a relocking conformational change and EF-G release from the ribosome complex. The resulting E 0 P 0 or E 02 P 02 (A-site unocupied) then moves on to the aa-tRNA selection step. Here, E 0 P 0 is the posttranslocational ribosome aligning with deX XXY (zero frame) and E 02 P 02 is the post-translocational ribosome aligning with cde XXX (-1 frame), where subscript 0 means a zero frame tRNA pairing with the zero frame; subscript 02 means a zero frame tRNA pairing with the À1 frame. E 0 P 0 may generate non-frameshift product NFS, or enter Pathway II or III described below. E 02 P 02 can generate frameshift product FS m , which incorporates the À1 frame aa-tRNA in the recoding site (YYY). In addition, it is also possible for E 02 P 02 to recruit a zero frame aa-tRNA for YYZ (A 0 ) and accommodate this aa-tRNA into the À1 frame. In this case, frameshift product FS z , which incorporates the zero frame aa-tRNA in the recoding site (YYZ), is produced (Pathway Ia).