One Sequence, Two Ribozymes: Implications for the Emergence of New Ribozyme Folds Erik A. Schultes and David P. Bartel

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Explanation of the ligation activity of truncated ribozymes.
For LIG2, LIG1, INT, and HDV1, the ligation products are represented by two bands in Fig. 2B (top panel), due to unanticipated ligation activity of truncated ribozyme molecules in addition to the activity of full-length RNA. On lower percentage gels, the faster-migrating product band resolves into seven distinct bands, corresponding to a loss of 9 to 15 nucleotides at the ribozyme 3´ terminus. These truncated ribozymes appear to adopt a class III-like fold as evidenced by the following: (i) They produce 2´-linkages. (ii) Nucleotide substitutions known to favor the class III ligase function and fold (C13A and C38G) lead to parallel increases in the activity of the truncated RNAs (Fig 2). (iii) Constructs LIG2, LIG1, INT, and HDV1 have fortuitous potential base pairing between segments corresponding to G51-U55 and A66-U70 in Fig. 2A. For the truncated molecules, this base pairing would lead to a fold that has all the class III features except the P4 stem-loop (Web fig. 2). The fortuitous potential pairing is not present in the original class III ligase isolates or in the constructs of the ligase neutral path (LIG4-LIG P in Fig. 3), explaining why only one product band was observed for LIG4-LIG P. (iv) The Pb(II)-cleavage pattern of the ligation product of truncated LIG2 was consistent with the formation of G51-U55:A66-U70 pairing, whereas probing of full-length LIG2, LIG1, and INT products confirmed formation of the anticipated P4 and P5 stems. None of the fortuitous pairs are present in the HDV secondary structure, and thus the alternative ligase fold, like the prototype ligase fold, has no base pairs in common with the HDV fold. The 3´ truncations contaminating the ribozyme preparations of LIG2, LIG1, INT, and HDV1 can generate over half of the ligated product (Fig. 2B), even though they compose only a minor fraction of the ribozyme preparation (presumably arising from RNA degradation or premature transcription termination). For these truncated molecules, a more optimal P5 might more than offset the loss of P4.

Supplemental Figure 2. The intersection sequence and a truncated form of this sequence assuming the class III ligase fold and the class III ligase-like fold, respectively.

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