Yeast lariat debranching enzyme. Substrate and sequence specificity.

K Nam,R.H Hudson,K.B Chapman,K Ganeshan,M.J Damha,J.D Boeke

doi:10.1016/s0021-9258(17)32037-9

Abstract

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.

Highlights

From the Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185,the ?Department of Chemistry, University of 'Ibronto, Ibronto, Ontario, Canada M5S lA4, and the Wepartment of Chemistry, McGill University, Montreal, Quebec, Canada H3A 2B2
Trinucleotide release assay using radiolabeled multicopy single-stranded DNAs (msDNAs) hDBR was originally identified by its ability to substrates was developed and used to determine the hbyadr-olyze the 2'-5'-phosphodiester bond in pre-mRNA-derived sicbiochemicalparameters-fortheenzyme.The de- RNA lariats and used to characterize the structure of lariat branching enzyme shows a strong preference for pu
We developed a yDBR assay system using msDNA Ec86 a s a substrate (Fig. 2). msDNA Ec86 labeled at the 5'phosphate was incubated with crude extractor partially purified yDBR, and the reaction products were analyzedon a 20% polyacrylamide, 8 sf urea gel (Fig. 3, lanes6-9 ).Amajor product with the approximatemobility of a trinucleotide wasobserved in the yDRR-treated lanes

Summary

11 To whom correspondence should be addressed

Dr Damha, Dept.of activity is mediated by a single polypeptide, whilemany steps inthe pre-mRNA splicing pathway require amultisubunit RNA-protein complex,the spliceosome. MsDNAs, as well as synthetic branched RNAs. DBR, debranching enzyme; yDBR, yeast DBR hDBR, human DBR. C ~ l i l 3 l 2 l f DE3) is a Iysogrn of a A drrivntivr which contains a singlr copy of T i RNA polymcrasr grnr undrr thr control of inducible promoter lor wrrr rr-purifirtlfrom a n X"; poly:lcrylamidr. Thr grl-purifird oligonuclrotidrs wrrr Inhrlrd lymrrasr which is undrrth rcontrol of lnc UV5 promoter. The productsof thr drhranchingrraclariat RNA was srparatcd from othrr species and purifird on an 8"; tion werr nnnlyzrd on an 8'; pnlyncrylamidr, 8 XI urrn grl for p ~ o u pI1 polyacrylamitlr. The self-splicing reaction was performrd for min a t 45 C in high salt huffer containing 500 my NH,tSO,),, 100 m\l \lfl12n,ntl 40 m\c Tris.HCl. h n r 1. The column was washed wit5hcolumn volumrs of huffrr A containing 250 m v KC1 and rlutrd with a 0.5 and 1.0 JI KC1 s t r p

RESULTS

DISCUSSION

UACUAC