The long D-stem of the selenocysteine tRNA provides resilience at the expense of maximal function.

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.

Active suppressor tRNAs with a double helix between the D- and T-loops.

One of the most conserved elements of the tRNA structure is the reverse-Hoogsteen base-pair T54--A58 in the T-loop, which plays a major role in the maintenance of the standard L-shape conformation. Here, we present the results of in vivo selection of 51 active suppressor tRNA clones, none of which contains base-pair T54--A58. In 49 clones, we found two regions in the D and T-loops that are complementary to each other. This finding suggests the existence of an inter-loop double helix consisting of three base-pairs, which could have the same role as base-pair T54--A58 in the fixation of the juxtaposition of the two helical domains within the L-shape. From this point of view, the appearance of the inter-loop double helix represents a compensatory effect for the absence of base-pair T54--A58. The results shed new light on the role of different elements of the tRNA structure in the formation of the standard L-shape conformation and on the possibility of synonymous replacements of one arrangement by another in functional RNA molecules.