RNase P ribozyme cleaves an RNA helix that resembles
the acceptor stem and T-stem structure of its natural ptRNA
substrate. When covalently linked with a guide sequence,
the ribozyme can function as a sequence-specific endonuclease
and cleave any target RNA sequences that base pair with
the guide sequence. Using a site-directed ultraviolet (UV)
cross-linking approach, we have mapped the regions of the
ribozyme that are in close proximity to a substrate that
contains the mRNA sequence encoding thymidine kinase of
human herpes simplex virus 1. Our data suggest that the
cleavage site of the mRNA substrate is positioned at the
same regions of the ribozyme that bind to the cleavage
site of a ptRNA. The mRNA-binding domains include regions
that interact with the acceptor stem and T-stem and in
addition, regions that are unique and not in close contact
with a ptRNA. Identification of the mRNA-binding site provides
a foundation to study how RNase P ribozymes achieve their
sequence specificity and facilitates the development of
gene-targeting ribozymes.