Abstract
BACKGROUND: The selenocysteine tRNA (tRNASec) has a uniquely long D-stem containing 6 base pairs. RESULTS: The extended D-stem is not essential for function but is required for stability. CONCLUSION: Enhanced secondary structure in selenocysteine tRNA compensates for the absence of canonical tertiary interactions. SIGNIFICANCE: The flexibility due to the absence of tertiary interactions is required for tRNASec function, whereas the enhanced secondary structure compensates for the decreased stability. The D-stem of the selenocysteine tRNA (tRNA(Sec)) contains 2 additional base pairs, which replace tertiary interactions 8-14 and 15-48 universally present in all other cytosolic tRNAs. To study the role of these additional base pairs in the tRNA(Sec) function, we used the instant evolution approach. In vivo screening of six combinatorial gene libraries provided 158 functional variants of the Escherichia coli tRNA(Sec). Analysis of these variants showed that the additional base pairs in the D-stem were not required for the tRNA(Sec) function. Moreover, at lower temperatures, these base pairs notably harmed the tRNA(Sec) activity. However, at elevated temperatures, these base pairs became essential as they made the tRNA structure more stable. The alternative way to stabilize the structure through formation of the standard tertiary interactions was not an option for tRNA(Sec) variants, which suggests that the absence of these interactions and the resulting flexibility of the tertiary structure are essential for tRNA(Sec) function.