2Institute of Genetics and Molecular and Cellular Biology (IGBMC), Department of Integrated Structural Biology, Illkirch, F-67404 France; fax: +33-3886-5320; INSERM, U596, Illkirch, F-67400 France; CNRS, UMR7104, Illkirch, F-67400 France; Université de Strasbourg, Strasbourg, F-67000 France
3Current address: Research and Education Center “Bionanophysics”, Moscow Institute of Physics and Technology, 141700 Dolgoprudnyi, Russia
4Current address: Institute of Genetics and of Molecular and Cellular Biology (IGBMC), Illkirch, F-67404 France
# These authors contributed equally to this work.
* To whom correspondence should be addressed.
Received December 17, 2012; Revision received January 24, 2013
In the process of protein synthesis, the translating ribosomes of eukaryotic cells form polyribosomes that are found to be multiplex functional complexes possessing elements of ordered spatial organization. As revealed by a number of electron microscopy studies, the predominant visible configurations of the eukaryotic polyribosomes are circles (circular polyribosomes) and two-stranded formations (so-called double-row polyribosomes). The “long” (i.e. heavy loaded) polyribosomes are usually represented by double-row structures, which can be interpreted as either topologically circular (“collapsed rings”), or topologically linear (zigzags or helices). In the present work we have analyzed the mRNA path within the eukaryotic polyribosomes, isolated from a wheat germ cell-free translation system, by integrating two approaches: the visualization of mRNA ends in polyribosomes by marking them with gold nanoparticles (3′-end) and initiating 40S subunits (5′-end), as well as by the cryoelectron tomography. Examination of the location of the mRNA markers in polyribosomes and mutual orientation of ribosomes in them has shown that the double-row polyribosomes of the same sample can have both circular and linear arrangements of their mRNA.
KEY WORDS: eukaryotic polyribosomes, mRNA, circular translation, cryoelectron tomography