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

Chimeric Lep proteins were generated by in vitro translation as was described previously. 51 Briefly, mRNA for each chimeric Lep protein was produced from plasmids using the RiboMAX RNA production system in accordance with the manufacturer's instructions (Promega, Madison, WI). Lep proteins were then produced from mRNA by in vitro translation using rabbit reticulocyte lysate (Promega, Madison, WI) supplemented with canine pancreatic rough microsomes (tRNA probes, College Station, TX) and EasyTag 35 S methionine (PerkinElmer, Waltham, MA). In vitro translation reactions were then diluted 1:4 into 1X SDS PAGE sample buffer and separated using a 12% SDS PAGE gel. To image radioactive translation products, PAGE gels were then dried, exposed to a phosphorimaging screen, and imaged using a Typhoon Imager (GE Healthcare, New York, NY). Image J software was then employed to process the data quantify the glycosylation state of each construct by densitometry.