Effects of 3′-terminal phosphates in RNA produced by ribozyme cleavage

Abstract

In vitro runoff transcription using T7 RNA polymerase has been the method of choice to produce milligram quantities of RNA for structural studies. Unfortunately, the T7 enzyme often adds one or more extra nucleotides at the 3′ end, which results in a heterogeneous RNA product (Milligan et al., 1987). This heterogeneity can be observed at the 5′ end as well, depending on the transcription template (Ferre-D'Amare & Doudna, 1996). The lack of homogeneity, which potentially is deleterious for structural studies, can be overcome with the use of cis- and/or trans-acting ribozymes, which produce clean RNA ends after their catalytic reaction (Ferre-D'Amare & Doudna, 1996). This procedure has been scaled up for large quantities of RNA for NMR and X-ray crystallographic studies, and is useful for purification of larger RNAs (greater than 50 nt) where single-nucleotide resolution by gel electrophoresis is difficult. Ribozymes that have been used in this manner include the hairpin ribozyme, the hammerhead ribozyme (HH), the hepatitis delta ribozyme (δ), and the Neurospora varkud satellite RNA ribozyme (VS) (Guo & Collins, 1995; Price et al., 1995; Ferre-D'Amare & Doudna, 1996). All of these ribozymes leave a 5′-hydroxyl and a 3′-cyclic phosphate as products of cleavage.