Abstract
In contrast to −1 programmed ribosomal frameshifting (PRF) stimulation by an RNA pseudoknot downstream of frameshifting sites, a refolding upstream RNA hairpin juxtaposing the frameshifting sites attenuates −1 PRF in human cells and stimulates +1 frameshifting in yeast. This eukaryotic functional mimicry of the internal Shine-Dalgarno (SD) sequence-mediated duplex was confirmed directly in the 70S translation system, indicating that both frameshifting regulation activities of upstream hairpin are conserved between 70S and 80S ribosomes. Unexpectedly, a downstream pseudoknot also possessed two opposing hungry codon-mediated frameshifting regulation activities: attenuation of +1 frameshifting and stimulation of a non-canonical −1 frameshifting within the +1 frameshift-prone CUUUGA frameshifting site in the absence of release factor 2 (RF2) in vitro. However, the −1 frameshifting activity of the downstream pseudoknot is not coupled with its +1 frameshifting attenuation ability. Similarly, the +1 frameshifting activity of the upstream hairpin is not required for its −1 frameshifting attenuation function Thus, each of the mRNA duplexes flanking the two ends of a ribosomal mRNA-binding channel possesses two functions in bi-directional ribosomal frameshifting regulation: frameshifting stimulation and counteracting the frameshifting activity of each other.
Highlights
Genetic codes and potential secondary structures defined by local RNA sequences constitute the two layers of information embedded within the primary mRNA sequences of an open reading-frame (ORF)
The GST open reading frame (ORF) was fixed in 0 frame, while the downstream Renilla luciferase gene (Rluc) ORF was fused out of frame such that it could only be accurately translated upon frameshifting through insertion of translational frameshifting signals between GST and Rluc ORFs (Figure S1A)
The −1 programmed ribosomal frameshifting (PRF) efficiencies induced by a downstream dnaX hairpin in the presence of upstream internal SD mediated duplexes with different spacings toward the frameshifting site (Figure S1B,D) were similar to those reported in vivo [12], indicating that the experimental platform can faithfully reproduce this −1 PRF model system
Summary
Genetic codes and potential secondary structures defined by local RNA sequences constitute the two layers of information embedded within the primary mRNA sequences of an open reading-frame (ORF). The secondary structures in mRNA are unwound by an intrinsic duplex-unwinding activity of ribosome during translation [1,2], allowing exposure of the buried genetic codes for decoding in the ribosomal A-site, while refolding co-translationally after leaving the ribosome. Specific mRNA structures could resist ribosomal unwinding and trigger backward slippage (toward the 5’-direction of mRNA) of the ribosome by one nucleotide within a slippery sequence to stimulate translational reading-frame switch toward the −1 frame. By positioning A- and P-site tRNAs over the slippery sequence while keeping Watson-Crick base-pairing identities of the codon-anticodon interactions at the first two positions of each codon unchanged in 0- and −1 frames, this configuration helps increase probability to slip the ribosome one nucleotide in the 5’ direction [5,6]. Specific mRNA signals can program a ribosome to slip forward
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