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In many archaeal tRNAs, archaeosine is found at position 15. During archaeosine biosynthesis, archaeal tRNA-guanine transglycosylase (ArcTGT) first replaces the guanine base at position 15 with 7-cyano-7-deazaguanine (preQ0). In this study, we investigated whether modified nucleosides in tRNA substrates would affect ArcTGT incorporation of preQ0. We prepared a series of hypomodified tRNAsSer(GGA) fromEscherichia colistrains lacking each tRNA-modifying enzyme. Measurement of ArcTGT kinetic parameters with the various tRNAsSer(GGA) as substrates showed that theKmdecreased due to the lack of modified nucleosides. The tRNAsSer(GGA) melting profiles resulted in experimental evidence showing that each modified nucleoside in tRNASer(GGA) enhanced tRNA stability. Furthermore, the ArcTGTKmstrongly correlated with the melting temperature (Tm), suggesting that the unstable tRNA containing fewer modified nucleosides served as a better ArcTGT substrate. These results show that preQ0 incorporation into tRNA by ArcTGT takes place early in the archaeal tRNA modification process.