The landscape of translational stall sites in bacteria revealed by monosome and disome profiling

Tomoya Fujita; Takeshi Yokoyama; Mikako Shirouzu; Hideki Taguchi; Takuhiro Ito; Shintaro Iwasaki

doi:10.1261/rna.078188.120

The landscape of translational stall sites in bacteria revealed by monosome and disome profiling

¹RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, Wako, Saitama 351-0198 Japan
²School of Life Science and Technology, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8503, Japan
³Laboratory for Protein Functional and Structural Biology, RIKEN Center for Biosystems Dynamics Research, Tsurumi-ku, Yokohama 230-0045, Japan
⁴Graduate School of Life Sciences, Tohoku University, Aoba-ku, Sendai 980-8577, Japan
⁵Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Midori-ku, Yokohama 226-8503, Japan
⁶Laboratory for Translation Structural Biology, RIKEN Center for Biosystems Dynamics Research, Tsurumi-ku, Yokohama 230-0045, Japan
⁷Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-8561, Japan

Corresponding author: shintaro.iwasaki{at}riken.jp

Abstract

Ribosome pauses are associated with various cotranslational events and determine the fate of mRNAs and proteins. Thus, the identification of precise pause sites across the transcriptome is desirable; however, the landscape of ribosome pauses in bacteria remains ambiguous. Here, we harness monosome and disome (or collided ribosome) profiling strategies to survey ribosome pause sites in Escherichia coli. Compared to eukaryotes, ribosome collisions in bacteria showed remarkable differences: a low frequency of disomes at stop codons, collisions occurring immediately after 70S assembly on start codons, and shorter queues of ribosomes trailing upstream. The pause sites corresponded with the biochemical validation by integrated nascent chain profiling (iNP) to detect polypeptidyl-tRNA, an elongation intermediate. Moreover, the subset of those sites showed puromycin resistance, presenting slow peptidyl transfer. Among the identified sites, the ribosome pause at Asn586 of ycbZ was validated by biochemical reporter assay, tRNA sequencing (tRNA-seq), and cryo-electron microscopy (cryo-EM) experiments. Our results provide a useful resource for ribosome stalling sites in bacteria.

Keywords

Received November 17, 2020.
Accepted November 24, 2021.

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