Regulation of Translation Elongation Revealed by Ribosome Profiling [Dataset_4]

Ribosomes undergo substantial conformational changes during translation elongation to accommodate incoming aminoacyl-tRNAs and translocate along the mRNA template. We used multiple elongation inhibitors and chemical probing to define ribosome conformational states corresponding to different sized ribosome-protected mRNA fragments (RPFs) generated by ribosome profiling. We show using various genetic and environmental perturbations that the previously identified 20-22 nucleotide (nt) RPFs correspond predominantly to ribosomes in a pre-accommodation state with an open 40S ribosomal A site while the classical 27-29 nt fragments correspond to ribosomes in a pre-translocation state with an occupied 40S ribosomal A site. These distinct ribosome conformational states revealed by ribosome profiling are seen in all eukaryotes tested including fungi, worms and mammals. This high-resolution ribosome profiling approach reveals the anticipated Rck2-dependent inhibition of translocation through eEF2 phosphorylation during hyperosmotic stress. These same approaches reveal a strong translation elongation arrest during oxidative stress where the ribosomes are trapped in a pre-translocation state, but in this case the translational arrest is independent of Rck2-driven eEF2 phosphorylation. These results provide new insights and approaches for defining the molecular events that impact translation elongation throughout biology. 24 biological samples are included for ribosome footprinting samples. These include HeLa, MB-MDA-231 and yeast cells and C. elegans embryos.