Ribosomal Pausing during Translation of an RNA Pseudoknot

The genomic RNA of the coronavirus infectious bronchitis virus contains an efficient ribosomal frameshift signal which comprises a heptanucleotide slippery sequence followed by an RNA pseudoknot structure. The presence of the pseudoknot is essential for high-efficiency frameshifting, and it has been suggested that its function may be to slow or stall the ribosome in the vicinity of the slippery sequence. To test this possibility, we have studied translational elongation in vitro on mRNAs engineered to contain a well-defined pseudoknot-forming sequence. Insertion of the pseudoknot at a specific location within the influenza virus PB1 mRNA resulted in the production of a new translational intermediate corresponding to the size expected for ribosomal arrest at the pseudoknot. The appearance of this protein was transient, indicating that it was a true paused intermediate rather than a dead-end product, and mutational analysis confirmed that its appearance was dependent on the presence of a pseudoknot structure within the mRNA. These observations raise the possibility that a pause is required for the frameshift process. The extent of pausing at the pseudoknot was compared with that observed at a sequence designed to form a simple stem-loop structure with the same base pairs as the pseudoknot. This structure proved to be a less effective barrier to the elongating ribosome than the pseudoknot and in addition was unable to direct efficient ribosomal frameshifting, as would be expected if pausing plays an important role in frameshifting. However, the stem-loop was still able to induce significant pausing, and so this effect alone may be insufficient to account for the contribution of the pseudoknot to frameshifting.