Spacer-length dependence of programmed-1 or-2 ribosomal frameshifting on a U6A heptamer supports a role for messenger RNA (mRNA) tension in frameshifting

Abstract

Programmed-1 ribosomal frameshifting is employed in the expression of a number of viral and cellular genes. In this process, the ribosome slips backwards by a single nucleotide and continues translation of an overlapping reading frame, generating a fusion protein. Frameshifting signals comprise a heptanucleotide slippery sequence, where the ribosome changes frame, and a stimulatory RNA structure, a stem-loop or RNA pseudoknot. Antisense oligonucleotides annealed appropriately 3′ of a slippery sequence have also shown activity in frameshifting, at least in vitro. Here we examined frameshifting at the U6A slippery sequence of the HIV gag/pol signal and found high levels of both-1 and-2 frameshifting with stem-loop, pseudoknot or antisense oligonucleotide stimulators. By examining-1 and-2 frameshifting outcomes on mRNAs with varying slippery sequence-stimulatory RNA spacing distances, we found that-2 frameshifting was optimal at a spacer length 1-2 nucleotides shorter than that optimal for-1 frameshifting with all stimulatory RNAs tested. We propose that the shorter spacer increases the tension on the mRNA such that when the tRNA detaches, it more readily enters the-2 frame on the U6A heptamer. We propose that mRNA tension is central to frameshifting, whether promoted by stem-loop, pseudoknot or antisense oligonucleotide stimulator. © 2012 The Author(s).