Polyribosomes of circular topology are prevalent in mammalian cells

Figure 1.. Cryo-ET analysis of a diluted HeLa lysate sample. Inserts represent the enlarged fragments with ribosome arrangements attributed to polysomes. Ring (R), double-row (DR) and indefinite shape (I) polyribosomes can be distinguished on the longitudinal section of the reconstructed tomogram. Arrows indicate flexible rRNA expansion segments (ES). Scale bars, 100 nm.

Figure 2.. Cryo-EM structure of translating HeLa 80S ribosome. (A) Result of subtomogram averaging. 3D tomogram fragments containing individual polysomal ribosomes were extracted, aligned, and averaged, giving the structure of translating ribosome at 17.6 Å resolution. The head of the 40S ribosomal subunit is dyed in red and the body is in yellow, the 60S subunit is in blue with a pink P1/P2 stalk. The mRNA entry and exit sites are indicated with red dotted lines, arrowheads indicate the 3′ to 5′ direction of the mRNA chain. (B) The crosscut showing the presence of A-site and P-site tRNA densities (hashed areas) in the averaged ribosome subtomograms. The dotted line marks the expected position of the absent E-site tRNA. (C) 7.7 Å structure of translating HeLa 80S ribosome obtained by conventional single particle cryo-EM analysis. The insert represents the crosscut slice visualizing the electron densities corresponding to A-site (green) and P-site (blue) tRNA models positioned in pre-translocation A-A and P-P states, respectively. No tRNA density was detected in the E-site (red model). tRNA models were fitted into the 80S map according to PDB 5AJ0 (50) for P- and E-site tRNAs and PDB 7NRC…

Figure 3.. Diversity of polysome organization in HeLa cell lysate. Examples of polyribosomes with circular (A), topologically linear zig-zag (B), helix-like (C), helical (D) and randomly shaped (E) ribosome arrangements. Copies of the 80S ribosome model acquired from subtomogram analysis were placed into the tomograms at positions and spatial orientations determined by the alignment procedure. Ribosome coloring is the same as in Figure 2A. Right panels: schemes of presumable mRNA pathways evaluated from relative orientation of ribosomes. Arrowheads indicate the direction of the mRNA chain in each ribosome, from the mRNA entry to the exit site.

Figure 4.. Moderate size polyribosomes of circular topology are common in HeLa cell lysates. Characteristic examples of circular polysomes with 40S subunits looking predominantly inside the ring. The point of view is chosen so that the ribosomes move clockwise along the mRNA chain.

Figure 5.. The average ribosome loading value (LV, Rs per mRNA) of circular polyribosomes is significantly less than those of the polysomes of linear topology. The box-plot represents the statistical spread of LV for circular and topologically linear (zig-zag, helical, helix-like and random shape) polyribosomes. The analysis included 93 circular and 164 linear polysomes.