title: Biochemical properties of Bacillus Calmette Guerin ribonuclease III.
abstract: Double-stranded RNA (dsRNA) is discovered to participate in the regulation of gene expression in both bacterial and eukaryotic cells. Members of ribonuclease III (RNase III) family recognize RNA motifs and cleave substrates at specific sites in a divalent-metal-ion-dependent manner. In this study, we find the RNase III from Bacillus Calmette Guerin (BCG-RNase III) cleaves small hairpin RNA based on the conserved stem structure associated with Mycobacterium 16S ribosomal RNA precursor at specific sites which are not determined. To evaluate the influence of remnant endogenous ribonucleases from expression host on RNA cleavage assays for RNase III, we use E44A and D48A mutant of the enzyme to perform RNA cleavage assays and find that remnant ribonucleases have no effect on cleavage assays. The RNA cleavage activity of the enzyme can be supported by Mg(2+), Mn(2+), and Co(2+) and enhanced with the increasing salt concentration. The catalytic activity of the enzyme is exhibited when the temperature of the reaction buffer ranges from 30 to 55 °C and the pH of the buffer from 7.0 to 10.0. Two major cleavage sites in RNA substrate are identified using RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE).
authors.affiliation: Tianjin Life Science Research Center and Basic Medical School, Tianjin Medical University, Tianjin, China.|Department of Clinical Laboratory, Tianjin First Center Hospital, Tianjin, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Yang
authors.affiliation: Tianjin Life Science Research Center and Basic Medical School, Tianjin Medical University, Tianjin, China.|Department of Clinical Laboratory, Tianjin Hospital of ITCWM Nankai Hospital, Tianjin, China.
authors.forename: Yan-Li
authors.initials: YL
authors.lastname: Wang
authors.affiliation: Tianjin Life Science Research Center and Basic Medical School, Tianjin Medical University, Tianjin, China.
authors.forename: Jin-Chuan
authors.initials: JC
authors.lastname: Dai
authors.affiliation: Tianjin Life Science Research Center and Basic Medical School, Tianjin Medical University, Tianjin, China.
authors.forename: Min
authors.initials: M
authors.lastname: Liu
authors.affiliation: Tianjin Life Science Research Center and Basic Medical School, Tianjin Medical University, Tianjin, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.affiliation: Tianjin Life Science Research Center and Basic Medical School, Tianjin Medical University, Tianjin, China.
authors.forename: Hua
authors.initials: H
authors.lastname: Tang
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D011487:Protein Conformation; D043244:Ribonuclease III / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D043244:Ribonuclease III
keywords: Bacillus Calmette Guerin; Enzyme catalysis; Hairpin RNA; RNase III; Ribosomal RNA