Gene silencing pathway RNA-dependent RNA polymerase of Neurospora crassa: yeast expression and crystallization of selenomethionated QDE-1 protein.
The RNA-dependent RNA polymerase, QDE-1, is a component of the RNA silencing pathway in Neurospora crassa. The enzymatically active carboxy-terminal fragment QDE-1 DeltaN has been expressed in Saccharomyces cerevisiae in the presence and absence of selenomethionine (SeMet). The level of SeMet incorporation was estimated by mass spectrometry to be approximately 98%. Both native and SeMet proteins were crystallized in space group P2(1) with unit cell parameters a=101.2, b=122.5, c=114.4A, beta=108.9 degrees , and 2 molecules per asymmetric unit. The native and SeMet crystals diffract to 2.3 and 3.2A, respectively, the latter are suitable for MAD structure determination.
Laurila
Minni R L
MR
Institute of Biotechnology, Faculty of Biosciences, Viikki Biocenter, University of Helsinki, P.O. Box 56, Viikinkaari 5, 00014 Helsinki, Finland.
Salgado
Paula S
PS
Makeyev
Eugene V
EV
Nettelship
Joanne
J
Stuart
David I
DI
Grimes
Jonathan M
JM
Bamford
Dennis H
DH
D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry*; D020868:Gene Silencing; D009492:Neurospora crassa / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012645:Selenomethionine
D005656:Fungal Proteins; D010446:Peptide Fragments; D012645:Selenomethionine; C119303:QDE1 protein, Neurospora crassa; D012324:RNA-Dependent RNA Polymerase