Sequence, structure, and function of hatchet ribozymes. (A) Updated consensus sequence and secondary structure model for hatchet ribozymes based on 210 different representatives. The arrowhead defines the site of ribozyme-mediated cleavage. Image was prepared using the R2R program (Weinberg and Breaker 2011). (B) Sequence and secondary structure of the bimolecular RNA construct Ht-1 based on metagenomic sequence SRS017191_Baylor_scaffold_14517/1281–1081 except that the loop region of P4 was replaced by UUCG. Nucleotides in red correspond to the most highly conserved positions in the consensus sequence. Nucleotides forming the 5′ and 3′ flanks of the cleavage site are numbered −1 and 1, respectively. Note that two non-native base pairs were added to P1 (brackets) to facilitate detection of the 3′ cleavage fragment by mass spectrometry. (C) Confirmation of ribozyme activity of Ht-1 carrying the two additional base pairs in P1. 5′ 32P-labeled substrate (∼5 nM) was incubated in the absence (−) or presence (+) of enzyme (∼100 nM) either in the absence or presence of 20 mM Mg2+ as indicated for each lane of the 20% PAGE gel. Additional details are described in Materials and Methods. The bands corresponding to the 16-nt substrate (sub) and the 8-nt 5′-cleavage product (5′-clv) are annotated accordingly. (D) Mass spectrometry analysis of a Ht-1 reaction depicting peaks corresponding closely with the calculated masses for 5′-clv and the corresponding 8-nt 3′-cleavage product annotated 3′-clv. The calculated (calc.) and observed (obs.) atomic masses for the cleavage products are presented. (E) Analysis of a Ht-1 construct with a 6-bp P1 and a 2′deoxyribonucleotide (dC) replacing the cytidine ribonucleotide (C) at position −1 of the substrate. Other annotations are as described in C.
