Direct three-dimensional localization and positive identification of RNA helices within the ribosome by means of genetic tagging and cryo-electron microscopy.
BACKGROUND
Ribosomes are complex macromolecular machines that perform the translation of the genetic message. Cryo-electron microscopic (cryo-EM) maps of the Escherichia coli 70S ribosome are approaching a resolution of 10 A and X-ray maps of the 30S and 50S subunits are now available at 5 A. These maps show a lot of details about the inner architecture of the ribosome and ribosomal RNA helices are clearly visible. However, in the absence of further biological information, even at the higher resolution of the X-ray maps many rRNA helices can be placed only tentatively. Here we show that genetic tagging in combination with cryo-EM can place and orient double-stranded RNA helices with high accuracy.


RESULTS
A tRNA sequence inserted into the E. coli 23S ribosomal RNA gene, at one of the points of sequence expansion in eukaryotic ribosomes, is visible in the cryo-EM map as a peripheral 'foot' structure. By tracing its acceptor-stem end, the location of helix 63 in domain IV and helix 98 in domain VI of the 50S subunit could be precisely determined.


CONCLUSIONS
Our study demonstrates for the first time that features of a three-dimensional cryo-EM map of an asymmetric macromolecular complex can be interpreted in terms of secondary and primary structure. Using the identified helices as a starting point, it is possible to model and interpret, in molecular terms, a larger portion of the ribosome. Our results might be also useful in interpreting and refining the current X-ray maps.
Spahn
C M
CM
Howard Hughes Medical Institute, Albany, NY, USA.
Grassucci
R A
RA
Penczek
P
P
Frank
J
J
D001483:Base Sequence; D020285:Cryoelectron Microscopy / Q000379:methods; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics* / Q000648:ultrastructure*; D007091:Image Processing, Computer-Assisted; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000235:genetics / Q000648:ultrastructure; D012338:RNA, Ribosomal, 23S / Q000235:genetics / Q000648:ultrastructure*; D012343:RNA, Transfer / Q000235:genetics / Q000648:ultrastructure*; D012270:Ribosomes / Q000648:ultrastructure*
D017931:DNA Primers; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D012343:RNA, Transfer