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Translation of mRNAs into proteins is a key cellular process. Ribosome binding sites and stop codons provide signals to initiate and terminate translation, while stable secondary mRNA structures can induce translational recoding events. Fluorescent proteins are commonly used to characterize such elements but require the modification of a part's natural context and allow only a few parameters to be monitored concurrently. Here, we combine Ribo-seq with quantitative RNA-seq to measure at nucleotide resolution and in absolute units the performance of elements controlling transcriptional and translational processes during protein synthesis. We simultaneously measure 779 translation initiation rates and 750 translation termination efficiencies across the Escherichia coli transcriptome, in addition to translational frameshifting induced at a stable RNA pseudoknot structure. By analyzing the transcriptional and translational response, we discover that sequestered ribosomes at the pseudoknot contribute to a σ32-mediated stress response, codon-specific pausing, and a drop in translation initiation rates across the cell. Our work demonstrates the power of integrating global approaches toward a comprehensive and quantitative understanding of gene regulation and burden in living cells.

Original publication

DOI

10.15252/msb.20188719

Type

Journal article

Journal

Mol Syst Biol

Publication Date

03/05/2019

Volume

15

Keywords

RNA‐seq, Ribo‐seq, genetic circuits, transcription, translation, Codon, Escherichia coli, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Genome, Bacterial, Nucleic Acid Conformation, Promoter Regions, Genetic, Protein Biosynthesis, RNA, Messenger, Ribosomes, Sequence Analysis, DNA, Sequence Analysis, RNA, Transcriptome