The cleavage step of ribonuclease P catalysis is determined by ribozyme-substrate interactions both distal and proximal to the cleavage site

The cleavage step of bacterial RNase P catalysis involves concentration-ind ependent processes after the formation of the ribozyme-substrate complex th at result in the breaking of a phosphodiester bond. The 2'OH group at the c leavage site of a pre-tRNA substrate is an important determinant in the cle avage step. We determined here that in contrast to a tRNA substrate, the 2' OH at the cleavage site of two in vitro selected substrates has no effect, whereas a 2'OH located adjacent to the cleavage site has a similarly large effect on the cleavage step. This result indicates that a unique 2'OH in th e vicinity of the cleavage site interacts with the ribozyme to achieve the maximal efficiency of the cleavage step. Individual modifications in a pre- tRNA substrate that disrupt ES interactions proximal to the cleavage site g enerally have little effect on the usage of this unique 2'OH. Ribozyme modi fications that delete the interactions involving the T stem-loop of the tRN A have a large effect on the usage of this unique 2'OH and also alter the l ocation of this 2'OH. We propose a new ES complex prior to the bond-breakin g step in the reaction scheme to explain these results. This second ES comp lex is in fast equilibrium with the initial ES complex formed by bimolecula r collision. The ribozyme interaction with this unique 2'OH shifts the equi librium in favor of the second ES complex. The formation of the second ES c omplex may require optimal geometry of the two independently folding domain s of this ribozyme to precisely position crucial functional groups and Mg2 ions in the active site. Such a domain geometry is significantly favored b y the RNase P protein. In the absence of the protein, spatial rearrangement of these domains in the ES complex may be necessary.