Recent structural studies provided detailed mechanistic insights of the ribosome in its different functional states and its interplay with ribosome-bound complexes involved in nascent peptide folding, processing or targeting. However, it is largely unknown how the translational machinery is spatially arranged in the context of translating polysomes. Here, cryo electron tomography and a template matching approach were employed to map 70S ribosomes in vitrified bacterial translation extracts and in lysates of active Escherichia coli spheroplasts. In these preparations, polysomal arrangements were observed in which neighboring ribosomes are densely packed and exhibit preferred orientations. The analysis of characteristic examples of polysomes reveals a staggered or pseudo-helical organization of ribosomes along the mRNA trace, with the transcript being sequestered on the inside, the tRNA entrance sites being accessible and the polypeptide exit sites facing the cytosol. Modeling of elongating nascent polypeptide chains suggests that this arrangement maximizes the distance between nascent chains on adjacent ribosomes, thereby reducing the probability of intermolecular interactions that would give rise to aggregation and limit productive folding. In a continuing study, we mapped individual ribosomes in electron-tomographic reconstructions of the periphery of intact human cells by template matching. The average structure of the human ribosome was determined at intermediate resolution. Interestingly, the relative spatial configuration of adjacent ribosomes in a cellular environment is also clearly non-random and preferred configurations are characteristic for polysomes. These configurations are comparable to those of bacterial polysomes and might hint to a conserved mechanism of self-organization to prevent nascent-chain interactions between adjacent ribosomes. This work should represent a further step towards the broader aim to comprehensively analyze the spatial distribution of macromolecules and the related biological significance within a close-to-native or even cellular context.     «

Recent structural studies provided detailed mechanistic insights of the ribosome in its different functional states and its interplay with ribosome-bound complexes involved in nascent peptide folding, processing or targeting. However, it is largely unknown how the translational machinery is spatially arranged in the context of translating polysomes. Here, cryo electron tomography and a template matching approach were employed to map 70S ribosomes in vitrified bacterial translation extracts...     »