EFFICIENT TRANS-CLEAVAGE OF A STEM-LOOP RNA SUBSTRATE BY A RIBOZYME DERIVED FROM NEUROSPORA VS RNA

We have constructed a ribozyme containing 144 nucleotides of Neurospora VS RNA that can catalyze the cleavage of a separate RNA in a true enzymatic manner (K-m approximate to 0.13 mu M, k(cat) approximate to 0.7/min). Comparison of the rates of cis- and trans-cleavage, as well as the lack of effect of pH on the rate of cleavage, suggest that a rate-limiting step, possibly a conformational change, occurs prior to cleavage. The minimum contiguous substrate sequence required for cleavage consists of one nucleotide upstream and 19 nucleotides downstream of the cleavage site. Unlike most other ribozymes which interact with long single-stranded regions of their substrates, the minimal substrate for the VS ribozyme consists mostly of a stable stem-loop, which would appear to preclude its recognition simply via extensive Watson-Crick base pairing.