A PHYSICAL ASSAY FOR AND KINETIC-ANALYSIS OF THE INTERACTIONS BETWEENM1 RNA AND TRANSFER-RNA PRECURSOR SUBSTRATES

A gel-shift assay was devised to detect stable enzyme-substrate (E-S) complexes between M1 RNA, the catalytic subunit of RNase P from Escher ichia coli, and its tRNA precursor substrates. The use of deletion der ivatives of M1 RNA in the gel-shift assay has allowed us to identify r egions of the enzyme that are involved in the binding of the substrate or that are necessary for catalytic activity. Fragments of substrates that contain the 3' CCA sequence bind preferentially to regions in th e 5' half of M1 RNA, while 5' leader sequences interact primarily with regions in the 3' half of M1 RNA. The 5' leader sequence present in t he precursor to tRNA(Tyr)su3 from E. coli plays an important role in t he formation of stable E-S complexes with M1 RNA. The CCA sequence at the 3' end of precursor tRNA substrates is involved in the product-rel ease step of the reaction that is catalyzed by M1 RNA. Direct measurem ents of the concentrations of all the components in the reaction catal yzed by M1 RNA facilitated a new approach to the kinetic analysis of t he action of the enzyme.