Over 73,000 projections of the E. coli ribosome bound with formyl-methionyl initiator tRNAf(Met) were used to obtain an 11.5 A cryo-electron microscopy map of the complex. This map allows identification of RNA helices, peripheral proteins, and intersubunit bridges. Comparison of double-stranded RNA regions and positions of proteins identified in both cryo-EM and X-ray maps indicates good overall agreement but points to rearrangements of ribosomal components required for the subunit association. Fitting of known components of the 50S stalk base region into the map defines the architecture of the GTPase-associated center and reveals a major change in the orientation of the alpha-sarcin-ricin loop. Analysis of the bridging connections between the subunits provides insight into the dynamic signaling mechanism between the ribosomal subunits.
Variations in the inner ribosomal landscape determining the topology of nascent protein transport have been studied by three-dimensional cryo-electron microscopy of erythromycin-resistant Escherichia coli 70S ribosomes. Significant differences in the mouth of the 50S subunit tunnel system visualized in the present study support a simple steric-hindrance explanation for the action of the drug. Examination of ribosomes in different functional states suggests that opening and closing of the main tunnel are dynamic features of the large subunit, possibly accompanied by changes in the L7/L12 stalk region. The existence and dynamic behavior of side tunnels suggest that ribosomal proteins L4 and L22 might be involved in the regulation of a multiple exit system facilitating cotranslational processing (or folding or directing) of nascent proteins.
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