title: Dimeric and monomeric Bacillus subtilis RNase P holoenzyme in the absence and presence of pre-tRNA substrates.
abstract: Ribonuclease P (RNase P) is a ribonucleoprotein enzyme that catalyzes the 5' maturation of tRNA precursors. The bacterial RNase P holoenzyme is composed of a large, catalytic RNA and a small protein. Our previous work showed that Bacillus subtilis RNase P forms a specific "dimer" that contains two RNase P RNA and two RNase P protein subunits in the absence of substrate. We investigated the equilibrium and the structure of the dimeric and the monomeric holoenzyme in the absence and presence of substrates by synchrotron small-angle X-ray scattering, 3' autolytic processing, and hydroxyl radical protection. In the absence of substrate, the dimer-monomer equilibrium is sensitive to monovalent ions and the total holoenzyme concentration. At 0.1 M NH4Cl, formation of the dimer is strongly favored, whereas at 0.8 M NH4Cl, the holoenzyme is a monomer. Primary hydroxyl radical protection in the dimer is located in the specificity domain, or domain I, of the RNase P RNA. The ES complex with a substrate containing a single tRNA is always monomeric. In contrast, the dominant ES complex with a substrate containing two tRNAs is dimeric at 0.1 M NH4Cl and monomeric at 0.8 M NH4Cl. Our results show that the B. subtilis holoenzyme can be a dimer and a monomer, and the fraction of the dimer is very sensitive to the environment. Under a variety of conditions, both the holoenzyme dimer and monomer can be present in significant amounts. Because the majority of tRNA genes are organized in large operons and because of the lack of RNase E in B. subtilis, a dimeric holoenzyme may be necessary to facilitate the processing of large precursor tRNA transcripts. Alternatively, the presence of two forms of the RNase P holoenzyme may be required for other yet unknown functions.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, 920 East 58th Street, Chicago, Illinois 60637, USA.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Barrera
authors.forename: Xingwang
authors.initials: X
authors.lastname: Fang
authors.forename: Jaby
authors.initials: J
authors.lastname: Jacob
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Casey
authors.forename: P
authors.initials: P
authors.lastname: Thiyagarajan
authors.forename: Tao
authors.initials: T
authors.lastname: Pan
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D001483:Base Sequence; D019281:Dimerization; D004722:Endoribonucleases / Q000737:chemistry*; D020035:Holoenzymes / Q000737:chemistry; D017665:Hydroxyl Radical / Q000737:chemistry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D012322:RNA Precursors / Q000737:chemistry*; D012329:RNA, Bacterial / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry*; D012343:RNA, Transfer / Q000737:chemistry*; D043262:Ribonuclease P; D012542:Scattering, Radiation; D013379:Substrate Specificity; D014965:X-Rays
chemical_list: D020035:Holoenzymes; D021122:Protein Subunits; D012322:RNA Precursors; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D017665:Hydroxyl Radical; D012343:RNA, Transfer; D004722:Endoribonucleases; D043262:Ribonuclease P