Actin filament labels for localizing protein components in large complexes viewed by electron microscopy

RNA. 2009 Feb;15(2):244-8. doi: 10.1261/rna.1313609. Epub 2008 Dec 17.

Abstract

Localizing specific components in three-dimensional reconstructions of protein complexes visualized in an electron microscope increases the scientific value of those structures. Subunits are often identified within the complex by labeling; however, unless the label produces directly visible features, it must be detected by computational comparison with unlabeled complex. To bypass this step, we generated a cloneable tag from the actin-nucleating protein Spire that produces a directly visible "pointer" to the subunit after actin polymerization. We have used this new label to identify the intron of the C complex spliceosome to its small domain by fusing the 10 kDa Spire moiety to the affinity label that binds recombinant stem loops in the pre-mRNA substrate and assembling an actin filament on the particle.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actin Cytoskeleton / chemistry*
  • Actins / chemistry
  • Affinity Labels / chemistry*
  • Animals
  • Bacterial Proteins / chemistry
  • Drosophila Proteins / chemistry*
  • Introns
  • Microfilament Proteins / chemistry*
  • Microscopy, Electron*
  • Multiprotein Complexes / chemistry*
  • Multiprotein Complexes / ultrastructure
  • Protein Subunits
  • RNA Precursors / chemistry*
  • Spliceosomes / chemistry
  • Thermotoga maritima / enzymology

Substances

  • Actins
  • Affinity Labels
  • Bacterial Proteins
  • Drosophila Proteins
  • Microfilament Proteins
  • Multiprotein Complexes
  • Protein Subunits
  • RNA Precursors
  • RuvB protein, Bacteria
  • spir protein, Drosophila