title: Local tertiary structure probing of ribonucleoprotein particles by nuclease fusion proteins.
abstract: Analyses of the conformational dynamics of the numerous cellular ribonucleoprotein particles (RNP) significantly contribute to the understanding of their modes of action. Here, we tested whether ribonuclease fusion proteins incorporated into RNPs can be used as molecular probes to characterize the local RNA environment of these proteins. Fusion proteins of micrococcal nuclease (MNase) with ribosomal proteins were expressed in S. cerevisae to produce in vivo recombinant ribosomes which have a ribonuclease tethered to specific sites. Activation of the MNase activity by addition of calcium led to specific rRNA cleavage events in proximity to the ribosomal binding sites of the fusion proteins. The dimensions of the RNP environment which could be probed by this approach varied with the size of the linker sequence between MNase and the fused protein. Advantages and disadvantages of the use of MNase fusion proteins for local tertiary structure probing of RNPs as well as alternative applications for this type of approach in RNP research are discussed.
authors.affiliation: Lehrstuhl für Biochemie III, Universität Regensburg, Regensburg, Germany.
authors.forename: Uli
authors.initials: U
authors.lastname: Ohmayer
authors.forename: Jorge
authors.initials: J
authors.lastname: Perez-Fernandez
authors.forename: Thomas
authors.initials: T
authors.lastname: Hierlmeier
authors.forename: Gisela
authors.initials: G
authors.lastname: Pöll
authors.forename: Lydia
authors.initials: L
authors.lastname: Williams
authors.forename: Joachim
authors.initials: J
authors.lastname: Griesenbeck
authors.forename: Herbert
authors.initials: H
authors.lastname: Tschochner
authors.forename: Philipp
authors.initials: P
authors.lastname: Milkereit
mesh_terms: D008836:Micrococcal Nuclease / Q000235:genetics / Q000378:metabolism; D062105:Molecular Docking Simulation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D059367:RNA Cleavage; D012335:RNA, Ribosomal / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D054683:Ribosome Subunits, Large, Eukaryotic / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
chemical_list: D012335:RNA, Ribosomal; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D012269:Ribosomal Proteins; D008836:Micrococcal Nuclease