Abstract
The selenocysteine tRNA (tRNASec) has a uniquely long D-stem containing 6 base pairs. The extended D-stem is not essential for function but is required for stability. Enhanced secondary structure in selenocysteine tRNA compensates for the absence of canonical tertiary interactions. 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.
Highlights
The selenocysteine tRNA has a uniquely long D-stem containing 6 base pairs
The alternative way to stabilize the structure through formation of the standard tertiary interactions was not an option for tRNASec variants, which suggests that the absence of these interactions and the resulting flexibility of the tertiary structure are essential for tRNASec function
General Approach—For analysis of the structure-function relationships in the E. coli tRNASec, we used the approach known as instant evolution [13, 17]
Summary
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 (tRNASec) 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 tRNASec function, we used the instant evolution approach. Among all cytosolic tRNAs, the tRNASec is distinguished by its unusual secondary structure of which the most documented feature is its long acceptor stem [2, 3]. □S This article contains supplemental Extended Experimental Procedure, Fig. S1, and Tables S1–S8
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