Yeast lariat debranching enzyme. Substrate and sequence specificity.
Yeast RNA lariat debranching enzyme has been purified to near homogeneity using a bacterial overproducer of the enzyme. The enzyme is capable of digesting a variety of branched nucleic acid substrates, including group II intron lariats, multicopy single-stranded DNAs (msDNAs), and a variety of synthetic branched RNAs. A trinucleotide release assay using radiolabeled msDNA substrates was developed and used to determine the basic biochemical parameters for the enzyme. The debranching enzyme shows a strong preference for purines at the 2'-position in both msDNA and synthetic branched RNA substrates, in accord with the structure of its native substrate, which always has a 2'-G residue. The use of small synthetic branched RNA substrates will allow systematic mechanistic and structural studies of this unique enzyme.
Nam
K
K
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185.
Hudson
R H
RH
Chapman
K B
KB
Ganeshan
K
K
Damha
M J
MJ
Boeke
J D
JD
D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007438:Introns; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D012326:RNA Splicing; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D009711:Nucleotides; D012313:RNA; D012316:RNA Nucleotidyltransferases; C048135:lariat debranching enzyme