Cryo-negative staining reveals conformational flexibility within yeast RNA polymerase I.

Détails

ID Serval
serval:BIB_28770
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Institution
Titre
Cryo-negative staining reveals conformational flexibility within yeast RNA polymerase I.
Périodique
Journal of molecular biology
Auteur⸱e⸱s
De Carlo S., Carles C., Riva M., Schultz P.
ISSN
0022-2836
Statut éditorial
Publié
Date de publication
2003
Volume
329
Numéro
5
Pages
891-902
Langue
anglais
Notes
Publication types: Journal Article ; Research Support, Non-U.S. Gov't - Publication Status: ppublish
Résumé
The structure of the yeast DNA-dependent RNA polymerase I (RNA Pol I), prepared by cryo-negative staining, was studied by electron microscopy. A structural model of the enzyme at a resolution of 1.8 nm was determined from the analysis of isolated molecules and showed an excellent fit with the atomic structure of the RNA Pol II Delta4/7. The high signal-to-noise ratio (SNR) of the stained molecular images revealed a conformational flexibility within the image data set that could be recovered in three-dimensions after implementation of a novel strategy to sort the "open" and "closed" conformations in our heterogeneous data set. This conformational change mapped in the "wall/flap" domain of the second largest subunit (beta-like) and allows a better accessibility of the DNA-binding groove. This displacement of the wall/flap domain could play an important role in the transition between initiation and elongation state of the enzyme. Moreover, a protrusion was apparent in the cryo-negatively stained model, which was absent in the atomic structure and was not detected in previous 3D models of RNA Pol I. This structure could, however, be detected in unstained views of the enzyme obtained from frozen hydrated 2D crystals, indicating that this novel feature is not induced by the staining process. Unexpectedly, negatively charged molybdenum compounds were found to accumulate within the DNA-binding groove, which is best explained by the highly positive electrostatic potential of this region of the molecule, thus, suggesting that the stain distribution reflects the overall surface charge of the molecule.
Mots-clé
Binding Sites, Contrast Media, Cryoelectron Microscopy, Crystallography, X-Ray, DNA, Imaging, Three-Dimensional, Models, Molecular, Protein Conformation, Protein Structure, Tertiary, RNA Polymerase I, RNA Polymerase II, Saccharomyces cerevisiae Proteins, Staining and Labeling
Pubmed
Web of science
Création de la notice
19/11/2007 12:26
Dernière modification de la notice
20/08/2019 13:07
Données d'usage