Metal dependence and branched RNA cocrystal structures of the RNA lariat debranching enzyme Dbr1.

ABSTRACT: Intron lariats are circular, branched RNAs (bRNAs) produced during pre-mRNA splicing. Their unusual chemical and topological properties arise from branch-point nucleotides harboring vicinal 2',5'- and 3',5'-phosphodiester linkages. The 2',5'-bonds must be hydrolyzed by the RNA debranching enzyme Dbr1 before spliced introns can be degraded or processed into small nucleolar RNA and microRNA derived from intronic RNA. Here, we measure the activity of Dbr1 from Entamoeba histolytica by using a synthetic, dark-quenched bRNA substrate that fluoresces upon hydrolysis. Purified enzyme contains nearly stoichiometric equivalents of Fe and Zn per polypeptide and demonstrates turnover rates of ∼3 s^-1 Similar rates are observed when apo-Dbr1 is reconstituted with Fe(II)+Zn(II) under aerobic

SUBMITTER: Clark NE 

PROVIDER: S-EPMC5187747 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature