An RNA substrate of 81 nucleotides was synthesized with the MAXIscript kit (Ambion) using T7 RNA polymerase. The RNA [22, 23] . GTP (grey sphere) initially binds to the apo-enzyme (structure 1) which promotes closure of the N-terminal nucleotidyl transferase (NT) domain and the C-terminal oligomer-binding (OB) fold domain (structure 3). This is followed by hydrolysis of the GTP substrate to produce the enzyme-GMP covalent intermediate (structure 4). Hydrolysis of GTP disrupts the interactions between the bound guanylate and the C-terminal OB fold domain, thus destabilizing the closed form of the enzyme, which opens up with the concomitant release of pyrophosphate (structure 5). This exposes the RNA-binding site of the enzyme (exact location unknown), thereby allowing the subsequent transfer of the GMP moiety onto the acceptor RNA (structure 7). The capped RNA is then released and the enzyme can reinitiate the pathway. doi:10.1371/journal.pone.0024806.g001 transcript was synthesized from the pBS-KSII+ plasmid (Stratagene) that had been linearized with HindIII. The RNA substrate was purified on a denaturing 20% polyacrylamide gel and visualized by ultraviolet shadowing. The corresponding band was excised and then eluted from the gel by an overnight incubation in 0.1% SDS and 0.5 M ammonium acetate. The RNA was then precipitated with ethanol and quantitated by spectrophotometry. Alternatively, radiolabeled RNA substrates were also synthesized by adding [a-32 P]ATP or [a-32 P]GTP to the transcription reaction.