title: Identification of protein structural elements responsible for the diversity of sequence preferences among Mini-III RNases.
abstract: Many known endoribonucleases select their substrates based on the presence of one or a few specific nucleotides at or near the cleavage site. In some cases, selectivity is also determined by the structural features of the substrate. We recently described the sequence-specific cleavage of double-stranded RNA by Mini-III RNase from Bacillus subtilis in vitro. Here, we characterized the sequence specificity of eight other members of the Mini-III RNase family from different bacterial species. High-throughput analysis of the cleavage products of Φ6 bacteriophage dsRNA indicated subtle differences in sequence preference between these RNases, which were confirmed and characterized by systematic analysis of the cleavage kinetics of a set of short dsRNA substrates. We also showed that the sequence specificities of Mini-III RNases are not reflected by different binding affinities for cognate and non-cognate sequences, suggesting that target selection occurs predominantly at the cleavage step. We were able to identify two structural elements, the α4 helix and α5b-α6 loop that were involved in target selection. Characterization of the sequence specificity of the eight Mini-III RNases may provide a basis for better understanding RNA substrate recognition by Mini-III RNases and adopting these enzymes and their engineered derivatives as tools for RNA research.
authors.affiliation: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, ul. Ks. Trojdena 4, 02-109 Warsaw, Poland.
authors.forename: Dawid
authors.initials: D
authors.lastname: Głów
authors.affiliation: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, ul. Ks. Trojdena 4, 02-109 Warsaw, Poland.
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Kurkowska
authors.affiliation: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, ul. Ks. Trojdena 4, 02-109 Warsaw, Poland.
authors.forename: Justyna
authors.initials: J
authors.lastname: Czarnecka
authors.affiliation: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, ul. Ks. Trojdena 4, 02-109 Warsaw, Poland.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Szczepaniak
authors.affiliation: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, ul. Ks. Trojdena 4, 02-109 Warsaw, Poland.
authors.forename: Dariusz
authors.initials: D
authors.lastname: Pianka
authors.affiliation: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, ul. Ks. Trojdena 4, 02-109 Warsaw, Poland.
authors.forename: Verena
authors.initials: V
authors.lastname: Kappert
authors.affiliation: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, ul. Ks. Trojdena 4, 02-109 Warsaw, Poland.
authors.forename: Janusz M
authors.initials: JM
authors.lastname: Bujnicki
authors.affiliation: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, ul. Ks. Trojdena 4, 02-109 Warsaw, Poland.
authors.forename: Krzysztof J
authors.initials: KJ
authors.lastname: Skowronek
mesh_terms: D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D059014:High-Throughput Nucleotide Sequencing; D059372:Nucleotide Motifs; D000072416:Protein Structural Elements; D059367:RNA Cleavage; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III / Q000737:chemistry* / Q000378:metabolism; D017423:Sequence Analysis, RNA; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D012367:RNA, Viral; D043244:Ribonuclease III