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 Neurospor a VS RNA that can catalyze the cleavage of a separate RNA in a true en zymatic 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 clea vage. The minimum contiguous substrate sequence required for cleavage consists of one nucleotide upstream and 19 nucleotides downstream of t he cleavage site. Unlike most other ribozymes which interact with long single-stranded regions of their substrates, the minimal substrate fo r the VS ribozyme consists mostly of a stable stem-loop, which would a ppear to preclude its recognition simply via extensive Watson-Crick ba se pairing.