Mutational analysis of the RNA pseudoknot component of a coronavirus ribosomal frameshifting signal

Abstract

The genomic RNA of the coronavirus IBV contains an efficient ribosomal frameshift signal at the junction of the overlapping 1a and 1b open reading frames. The signal is comprised of two elements, a heptanucleotide "slip-site" and a downstream tertiary RNA structure in the form of an RNA pseudoknot. We have investigated the structure of the pseudoknot and its contribution to the frameshift process by analysing the frameshifting properties of a series of pseudoknot mutants. Our results show that the pseudoknot structure closely resembles that which can be predicted from current building rules, although base-pair formation at the region where the two pseudoknot stems are thought to stack co-axially is not a pre-requisite for efficient frameshifting. The stems, however, must be in close proximity to generate a functional structure. In general, the removal of a single base-pair contact in either stem is sufficient to reduce or abolish frameshifting. No primary sequence determinants in the stems or loops appear to be involved in the frameshift process; as long as the overall structure is maintained, frameshifting is highly efficient. Thus, small insertions into the pseudoknot loops and a deletion in loop 2 that reduced its length to the predicted functional minimum did not influence frameshifting. However, a large insertion (467 nucleotides) into loop 2 abolished frameshifting. A simple stem-loop structure with a base-paired stem of the same length and nucleotide composition as the stacked stems of the pseudoknot could not functionally replace the pseudoknot, suggesting that some particular conformational feature of the pseudoknot determines its ability to promote frameshifting.