title: Analysis of substrate recognition by the ribonucleoprotein endonuclease RNase P.
abstract: Ribonuclease P (RNase P), is a ribonucleoprotein complex that catalyzes the site-specific cleavage of pre-tRNA and a wide variety of other substrates. Although RNase P RNA is the catalytic subunit of the holoenzyme, the protein subunit plays a critical role in substrate binding. Thus, RNase P is an excellent model system for studying ribonucleoprotein function. In this review we describe methods applied to the in vitro study of substrate recognition by bacterial RNase P, covering general considerations of reaction conditions, quantitative measurement of substrate binding equilibria, enzymatic and chemical protection, cross-linking, modification interference, and analysis of site-specific substitutions. We describe application of these methods to substrate binding by RNase P RNA alone and experimental considerations for examining the holoenzyme. The combined use of these approaches has shown that the RNA and protein subunits cooperate to bind different portions of the substrate structure, with the RNA subunit predominantly interacting with the mature domain of tRNA and the protein interacting with the 5(') leader sequence. However, important questions concerning the interface between the two subunits and the coordination of RNA and protein subunits in binding and catalysis remain.
authors.affiliation: Center for RNA Molecular Biology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
authors.forename: Eric L
authors.initials: EL
authors.lastname: Christian
authors.forename: Nathan H
authors.initials: NH
authors.lastname: Zahler
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Kaye
authors.forename: Michael E
authors.initials: ME
authors.lastname: Harris
mesh_terms: D004722:Endoribonucleases / Q000737:chemistry / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012322:RNA Precursors / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D043262:Ribonuclease P; D013379:Substrate Specificity
chemical_list: D012322:RNA Precursors; D016337:RNA, Catalytic; D004722:Endoribonucleases; D043262:Ribonuclease P