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. 2018 Nov;10(11):a032797. doi: 10.1101/cshperspect.a032797

Figure 2.

Figure 2.

Regulation of self-splicing ribozyme function by a riboswitch aptamer. (A) Schematic representation of the 5′ untranslated region (5′UTR) of the CD3246 gene from Clostridium difficile, which carries a c-di-GMP-II riboswitch aptamer immediately upstream of a group I self-splicing ribozyme. The binding of c-di-GMP to the aptamer domain requires the structure depicted. This structure promotes ribozyme-catalyzed attack of GTP (called GTP1) at the 5′ splice site (5′SS) and subsequent attack of the 5′ exon’s 3′ terminus oxygen on the phosphorus of the 3′ splice site (3′SS) to yield a properly spliced messenger RNA (mRNA) product that can be translated. In the absence of c-di-GMP binding, portions of the aptamer and ribozyme shaded in blue reorganize to form an alternative base-paired structure. This disrupts the P1 stem that is required for proper splicing, and permits the formation of P1* to present an alternative site for GTP attack (called GTP2). GTP attack at this alternative site removes all but four nucleotides located upstream of the unusual UUG start codon for the ORF. (B) Two reaction products produced by the tandem aptamer-ribozyme construct depicted in A. (Top) Processed mRNA product generated by ribozyme action in the absence of c-di-GMP binding. Note that the ribosome-binding site (RBS) has been removed, and thus translation of the downstream open reading frame (ORF) is precluded. (Bottom) Processed mRNA product resulting from ribozyme splicing in the presence of c-di-GMP. Splicing creates a strong RBS that permits translation to occur. (C) Although the properly spliced mRNA (B, bottom) carries an RBS, the riboswitch aptamer presumably can still operate to occlude or reveal this sequence based on the availability of c-di-GMP. Specifically, the nucleotides shaded in blue that alternatively pair to regulate ribozyme splicing activity remain present in the properly spliced mRNA, and presumably can be exploited as an expression platform to continue to regulate gene expression as would a more common riboswitch.