Dissecting the energetic architecture within an RNA tertiary structural motif via high-throughput thermodynamic measurements

Structured RNAs and RNA/protein complexes perform critical cellular functions. They often contain structurally conserved tertiary contact motifs, whose occurrence simplifies the RNA folding landscape. Prior studies have focused on the conformational and energetic modularity of intact motifs. Here, we turn to the dissection of one common motif, the 11nt receptor (11ntR), using quantitative analysis of RNA on a massively parallel array to measure the binding of all single and double 11ntR mutants to GAAA and GUAA tetraloops, thereby probing the energetic architecture of the motif. While the 11ntR behaves as a motif, its cooperativity is not absolute. Instead, we uncovered a gradient from high cooperativity amongst base-paired and neighboring residues to additivity between distant residues. As expected, substitutions at residues in direct contact with the GAAA tetraloop resulted in the largest decreases to binding, and energetic penalties of mutations were substantially smaller for binding to the alternate GUAA tetraloop, which lacks tertiary contacts present with the canonical GAAA tetraloop. However, we found that the energetic consequences of base partner substitutions are not, in general, simply described by base pair type or isostericity. We also found exceptions to the previously established stability-abundance relationship for 11ntR sequence variants. These findings of exceptions to the rule highlight the power of systematic high-throughput approaches to uncover novel variants for future study in addition to providing an energetic map of a functional RNA.

Automated summary

Structured RNAs and RNA/protein complexes play critical roles in cellular functions. This study focuses on dissecting the 11nt receptor (11ntR) motif to understand its energetic architecture by measuring the binding of single and double mutants to tetraloops. The findings reveal a gradient of cooperativity and exceptions to the stability-abundance relationship, highlighting the power of high-throughput approaches.