Models for the mechanism of internal U-incorporation.
The experimentally determined stereoconfigurations (RP and SP) of reactive phosphorothioates are indicated. The compatibility of the experimental data with the various models is discussed in the text. (A) Original enzyme cascade model: The incorporated U residues are derived directly from UTP. (B) Transesterification model and (C) modified enzyme cascade model: The uridines are incorporated via the gRNA U-tail; chimeric gRNA-mRNA molecules are obligatory reaction intermediates. Incorporation occurs either via two successive transesterification reactions in (B), or via two rounds of cleavage-ligation in (C). Incorporation of only one uridine at one site is shown for each model. The site of U-incorporation in the mRNA is shown between an A and a G residue.