Figure 3.

Reduced +1 frameshifting by a downstream pseudoknot coexists with an unusual frameshifting event in the absence of RF2. (A) Sequences of the constructs designed to evaluate +1 attenuation activity of downstream mPK pseudoknot variants. MT and SL1 are the variants of mPK with mutations (in colors) to destabilize the stem1 and stem2, respectively. Please note that there are three UAA stop codons (with different colors) to terminate translation in three different frames in the absence of RF2 in these constructs using the pET GST-Rluc-1 reporter (Figure S1A) as the backbone. The −1 and +1 frame UAA stop codons are located in the N-terminal region of Rluc ORF with the −1 frame stop codon being moved to downstream of +1 frame stop codon in the “shift” construct. (B) The +1 frameshifting attenuation assays by SDS-PAGE analysis of ³⁵S methionine-labeled in vitro translation products for different mPK variant constructs in (A), with +1 PRF and potential −1 PRF products annotated. Calculated −1 and +1 PRF efficiency values displayed are means ± SD of three independent experiments. (C) Relative +1 PRF activity of construct with downstream WT mPK compared to MT with asterisks indicating p values < 0.05 using Student’s t-test.