Abstract: Little is known about the role of specific base modifications of transfer RNAs. Wyosine bases (Ybs) are tRNAPhe-specific modifications that are distinguished by differentiated, lateral side chains and base methylations appended to the core ring structure of a universally conserved G37, adjacent to the anticodon of Phe tRNAs. Based on previous data, we hypothesized that this modification was needed for -1 frameshifting. Using a reporter system incorporating a SCV-LA yeast virus slippery site for detecting -1 frameshifts in vivo, yeast strains were created that enabled chemical-genetic dissection of the role of different functional groups of wyebutosine (yW) that are added in a 3-step post-transcriptional set of reactions. With this system, hypomodification increased Phe-specific frameshifting, with incremental changes in frameshift efficiency following specific intermediates in the progression of yW synthesis. These data, combined with investigations of wild-type and hypomodified tRNA binding to ribosomes, suggest that frameshift efficiency is kinetically and not thermodynamically controlled. The progressive nature of frameshift efficiency with stage of modification is consistent with a step-wise evolution and tuning of frameshift potential. The stepwise tuning of frameshift efficiency could explain why tRNAPhe in some eukaryotes is not fully modified, but rather hypomodified to capture a specific frameshift potential.