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

The average mKate intensity among cells expressing a reporter construct bearing mutations that knock out the native ribosomal slip-site (UUUUUUA→ GUUCCUA, SSKO) is 79 ± 5% lower than that that among cells expressing the WT form of the reporter (n = 3, Fig. 4A ), which confirms that mKate intensities reflect the efficiency of -1PRF. The EE substitutions in TM2 decrease mKate intensity by 61 ± 16% relative to WT (n = 3, Fig. 4A ). In contrast, the LL substitutions increase the mKate intensity by 30 ± 11% (n = 3, Fig. 4A ). It should be emphasized that each of these reporters contain both the native slip-site and stem loop regions, and that these mutations do not alter their sequences. Thus, these findings demonstrate that -1PRF is sensitive to mutations that impact the membrane integration efficiency of TM2. Together, biochemical evidence suggests that TM2 is inefficiently recognized by the translocon (Fig. 1E) , and cellular topology reporters suggest that this segment is most often localized within the cytosol (Fig. 2) . Nevertheless, mutagenesis reveals that the propensity of the nascent chain to form a secondary topomer is positively correlated with -1PRF. These results are therefore suggestive of a mechanistic link between topogenesis and -1PRF.