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.

*To whom correspondence should be addressed. Tel: [phone]; Fax: [phone]; Email: [e-mail] Three major models have been proposed for the mechanism of À1 PRF ( Figure 1 ). One hypothesis proposes that À1 PRF takes place during accommodation of the aa-tRNA (8, 15, 16) . We have denoted this Pathway II. The simultaneous-slippage model (8) originally suggested that peptidyl-and aa-tRNAs simultaneously slip by one base in the 5 0 -direction to base pair with the À1 frame codons in the slippery site. In a refinement of this model (15) , À1 PRF was posited to occur when aa-tRNA and peptidyl-tRNA are located in the A/T entry and P/P site. The 9-Å model of À1 PRF (16) built upon both this and newly available structural data to propose that the $9-Å movement of the anticodon loop in the 5 0 -direction during aa-tRNA accommodation is constrained by the presence of the downstream stimulatory RNA structural element. This creates tension on the mRNA between the decoding center and the stimulatory element that can be relieved by decoupling of the A-and P-site tRNAs from the mRNA followed by subsequent slippage of the mRNA by one base in the 3 0 -direction relative to the tRNAs, resulting in a net slip reading frame by À1 base. Consistent with this model, mutations altering aa-tRNA accommodation were found to affect À1 PRF (18) (19) (20) . However, the simultaneous slippage-based models do not explain the role of sequences upstream of the slippery site, which have also been shown to affect the À1 PRF efficiency (21, 22) . A second general hypothesis proposes that À1 PRF occurs during translocation. This can be modeled through two discrete kinetic pathways. The first suggested that after peptidyltransfer, the two tRNAs move to P/E and A/P states, followed by an incomplete, two-base translocation event promoted by the downstream mRNA stimulatory structure (23) . During this incomplete translocation event, the tRNAs dissociate from the mRNA and re-pair with the À1 frame codons in the slippery site. We call this Pathway III. In support of this model, cryoelectron microscopy imaging revealed that a À1 PRF stimulating pseudoknot can interact with the ribosome to block the mRNA entrance channel, compromising the translocation process during À1 PRF (24) . The second co-translocational model proposed that incomplete translocation occurs one elongation cycle prior to the model by Weiss et al. (23) , and that tRNAs in the ribosomal E-, P-and A-sites are all involved in the process (22) . This model suggests that incomplete translocation promotes formation of a transition intermediate, and that entry of the new aa-tRNA into the ribosome and the tendency of tRNAs to revert to stable states drives the shift in reading frame. This is Pathway I. This model is supported by the demonstration that mutations altering E-site tRNA binding affect À1 PRF (22) . However, Figure 1 . A mechanistic model of -1 programmed ribosomal frameshifting. Two translation elongation cycles are depicted at the top: the ribosome undergoes decoding (DC), aa-tRNA accommodation (AA), peptidyltransfer (PT) and translocation (TL) twice to add two amino acids into the polypeptide sequences. A shift in reading frame may occur at the first TL step and the ribosome decodes a À1 frame A-site codon at the recoding site. Additionally, À1 PRF may occur during the second AA step, in which the ribosome has decoded the zero frame A-site codon. Incorporation of the À1 reading frame aa-tRNA s