Bacterial ribosome pause sites surveyed by an integration of ribosome profiling and nascent chain profiling II

Ribosome pauses are associated with diverse co-translational events and determine the fate of mRNAs and proteins. Thus the identification of the precise pause sites across transcriptome is a key, however, the landscape in bacterial has remained ambiguous. Here, we harnessed the multiple ribosome profiling strategies (standard, high-salt-wash, and disome) to survey the robust ribosome pause sites in E. coli. The found pause sites showed the correspondence with biochemical validation by integrated nascent chain profiling (iNP), which detects polypeptidyl-tRNA, an elongation intermediate. Among the list, ribosome pause at Asn586 of ycbZ was ensured by biochemical reporter assay, tRNA-seq, and cryo-electron microscopy. Our results provide a useful resource of ribosome stalling sites in bacteria. Overall design: Ribosome profiling of monosome is mono-ribosome (we analyzed ribosome footprints derived from one ribosome in the library), and disome is di-ribosome (we analyzed ribosome footprints derived from collied ribosomes in the library). Both samples were frozen in liquid nitrogen.