Dr. Julio Ortiz
Max Plank Institute of Biochemistry
Germany

Monday, December 13, 2010
3:00 pm (CT)
3269 Beckman Institute

Abstract

Cryoelectron tomography (CET) allows the visualization of flexible molecular structures both in vitro and in situ, i.e., in the functional environment of intact cells. To illustrate the opportunities provided by CET, two examples of spatial arrangements of ribosomes will be discussed in some detail, the native 3D organization of Escherichia coli ribosomes in polysomes and hibernating ribosomes (100S). We have applied CET to: a) E. coli lysates translating truncated mRNA to favor polysome formation; b) ribosome-enriched fractions of starved E. coli cells and c) intact E. coli cells grown in several conditions. We track the localization and orientation of ribosomes within tomograms using a known structure by template matching. Then, we align subtomograms containing single ribosomal particles to a common origin and average them to reveal details of the interaction between the identified complexes [1,2]. We showed that E. coli ribosomes adopt two preferential relative orientations in densely-packed polysomes. These alternative manners of ribosomal pairing result in variable 3D polysomal organizations, i.e, pseudo-planar or pseudo-helical polysomes [2]. It was also possible to purify in silico a particular ribosomal arrangement of the two 70S ribosomes that form a dimer in starvation conditions (100S ribosomes). Moreover, this 100S ribosomal arrangement has been detected in tomograms of intact E. coli cells only in stationary phase. We found distinctive spatial organization for translating and hibernating ribosomes. We hypothesize that polysomal organizations disfavor interaction between the non-folded nascent chains avoiding protein misfolding; whereas ribosome dimerization might protect the ribosomal subunits from degradation. In situ detection of the 100S ribosomes reinforces their physiological role as a storage form of ribosomes important for cell survival.