Cotranslational Folding Stimulates Programmed Ribosomal Frameshifting in the Alphavirus Structural Polyprotein

Haley R. Harrington; Matthew H. Zimmer; Laura M. Chamness; Veronica Nash; Wesley D. Penn; Thomas F. Miller; Suchetana Mukhopadhyay; Jonathan P. Schlebach

To produce a series of reporter constructs for ribosomal frameshifting, Gibson assembly was used to replace the portion of the polyprotein gene that falls 100 basepairs downstream from the ribosomal slips site in the 6K gene with a cassette containing an mKate gene in the -1 reading frame followed by an IRES and a GFP gene (Supplemental Fig. 4 ). The frameshift reporter (mKate) was fused 100 nucleotides downstream from the slip site in order to avoid disrupting the stimulatory RNA hairpin downstream from the slip sites. 22, 23 In addition to avoiding potential issues related to the impact of fusion domains on SRP-targeting of the nascent chain, this design also avoids recently described artefacts associated with previous generations of the dual-luciferase reporter system in two ways. 39 The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/790444 doi: bioRxiv preprint