Gene silencing pathway RNA-dependent RNA polymerase of Neurospora crassa: yeast expression and crystallization of selenomethionated QDE-1 protein

J Struct Biol. 2005 Jan;149(1):111-5. doi: 10.1016/j.jsb.2004.10.001.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cloning, Molecular
  • Crystallization
  • Crystallography, X-Ray*
  • Fungal Proteins / chemistry*
  • Gene Silencing
  • Neurospora crassa / enzymology*
  • Peptide Fragments / chemistry
  • RNA-Dependent RNA Polymerase / chemistry*
  • RNA-Dependent RNA Polymerase / genetics
  • Saccharomyces cerevisiae / genetics
  • Selenomethionine

Substances

  • Fungal Proteins
  • Peptide Fragments
  • Selenomethionine
  • QDE1 protein, Neurospora crassa
  • RNA-Dependent RNA Polymerase