The track of the pre-tRNA 5 ' leader in the ribonuclease P ribozyme-substrate complex

The ribonuclease P (RNase P) ribozyme is an endonuclease that binds precurs or tRNAs and catalyzes the removal of 5' leader nucleotides. Biochemical an d photo-cross-linking studies have identified sites of contact between the mature tRNA domain of pre-tRNA and the ribozyme; however, relatively little is known about the location of the 5' leader in the ribozyme-substrate com plex. To investigate the local three-dimensional environment of the 5' lead er, we employed the short-range photo-cross-linking agent 4-thiouridine (s( 4)U). The s(4)U photoagent was incorporated into a series of pre-tRNA subst rates containing unique uridine residues in the 5' leader sequence at posit ions -1, -3, -5, -7, or -10. The modified substrates formed high-affinity c omplexes with the ribozyme and produced discrete intermolecular cross-links to RNase P RNA from Bacillus subtilis. Locations of the cross-linked nucle otides in the ribozyme and pre-tRNA were determined by reverse transcriptas e primer extension. Photoagents incorporated into the 5' leader detected di screte elements of ribozyme structure in a progression from J18/2 to L15 to P3. Importantly, all of the cross-linked species retained the ability to c leave the covalently attached pre-tRNA, indicating that the cross-links ref lect the native structure of the ribozyme-substrate complex. Together with available structural and biochemical data, the cross-linking results sugges t a model for the position of the 5' leader within the ground-state ribozym e-substrate complex.