RNA sequence to structure analysis from comprehensive pairwise mutagenesis of multiple self-cleaving ribozymes

Abstract

Self-cleaving ribozymes are RNA molecules that catalyze the cleavage of their own phosphodiester backbones. These ribozymes are found in all domains of life and are also a tool for biotechnical and synthetic biology applications. Self-cleaving ribozymes are also an important model of sequence to function relationships for RNA because their small size simplifies synthesis of genetic variants and self-cleaving activity is an accessible readout of the functional consequence of the mutation. Here we used a high-throughput experimental approach to determine the relative activity for every possible single and double mutant of five self-cleaving ribozymes. From this data, we comprehensively identified non-additive effects between pairs of mutations (epistasis) for all five ribozymes. We analyzed how changes in activity and trends in epistasis map to the ribozyme structures. The variety of structures studied provided opportunities to observe several examples of common structural elements, and the data was collected under identical experimental conditions to enable direct comparison. Heat-map based visualization of the data revealed patterns indicating structural features of the ribozymes including paired regions, unpaired loops, non-canonical structures and tertiary structural contacts. The data also revealed signatures of functionally critical nucleotides involved in catalysis. The results demonstrate that the data sets provide structural information similar to chemical or enzymatic probing experiments, but with additional quantitative functional information. The large-scale data sets can be used for models predicting structure and function and for efforts to engineer self-cleaving ribozymes.

Discovery ofThetaRibozymes in Gut Phages–Implications for tRNA and Alternative Genetic Coding

“…Two pK a values were inferred for each construct: pK a1 ≈ 9.0 probably corresponding to a hydrated Mg 2+ ion and pK a2 ≈ 6.0 related to the catalytic cytosine residue (table S3). Furthermore, we confirmed the inactivation of all tRNA/Θrz pairs upon mutating the catalytic cytosine to uracil (CΔU), in line with known HDV-like behavior (29). This observation further validates our approach for identifying false-positives in the bioinformatic search (Fig.…”

“…We chose eight recently annotated viral databases from diverse environments (table S1) and cross-referenced all subsequent hits with tRNA motif searches in the same databases. To increase motif specificity, we incorporated false-positive motifs as internal controls, considering previous findings that drzs are inactivated by a cytosine-to-uracil mutation (CΔU) at the active site (28), with no observed rescue mutation at this position (29). Each search was conducted with four different descriptor files, one active ribozyme motif with the catalytic cytosine residue intact (first position in the J4/2 junction) and three false-positive motifs containing substitutions at this residue (CΔA, CΔG, and CΔU, respectively; Fig.…”

Identification of HDV-like theta ribozymes involved in tRNA-based recoding of gut bacteriophages

“…We chose eight recently annotated viral databases 23 , 29 – 35 from diverse environments (Supplementary Table 1 ) and cross-referenced all subsequent hits with tRNA motif searches in the same databases. To increase motif specificity, we incorporated false-positive motifs as internal controls, considering previous findings that drzs are inactivated by a cytosine-to-uracil mutation (CΔU) in the active site 36 , with no observed rescue mutation at this position 37 . Each search was conducted with four different descriptor files, one active ribozyme motif with the catalytic cytosine residue intact (first position in the J4/2 junction) and three false-positive motifs containing substitutions at this residue (CΔA, CΔG, and CΔU, respectively; Fig.…”

Flanking Sequence Cotranscriptionally Regulates Twister Ribozyme Activity

“…10,[13][14][15]17,18 Mutational studies have demonstrated that changes to the general acid and general base are not well-tolerated. [12][13][14]17,19 The reaction can also be assisted by buffer catalysis under biological reaction conditions. 20 Additionally, ∼90% of the twister ribozymes that were initially predicted by Breaker and co-workers possess one or more auxiliary stem-loops, referred to as P3 and P5 (Figure 1A).…”

“…Twister ribozymes have been studied extensively, and many of their structural and biochemical properties have been elucidated. , Structurally, the catalytic core of the twister ribozyme is comprised of three essential pairings, P1, P2, and P4 (Figure A), although variants suggest that P1 is dispensable for self-cleavage. ,− The cleavage site is located between residues −1 and 1 in L1, where residue −1 can be any nucleotide and residue 1 is a conserved A . Self-cleavage occurs via a general acid–base mediated mechanism, in which A1 acts as the general acid and the first G on the 3′ strand of L1 acts as the general base. ,− ,, Mutational studies have demonstrated that changes to the general acid and general base are not well-tolerated. − ,, The reaction can also be assisted by buffer catalysis under biological reaction conditions . Additionally, ∼90% of the twister ribozymes that were initially predicted by Breaker and co-workers possess one or more auxiliary stem-loops, referred to as P3 and P5 (Figure A) .…”