Absolute quantification of translational regulation and burden using combined sequencing approaches
Thomas E. Gorochowski, Irina Chelysheva, Mette Eriksen, Priyanka Nair, Steen Pedersen, Zoya Ignatova
Received: 4th June 18
Translation of mRNAs into protein 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, commonly used to characterize such elements, require the modification of a part’s natural context and allow only a few parameters to be monitored concurrently. Here, we develop a methodology that combines ribosome profiling (Ribo-seq) with quantitative RNA sequencing (RNA-seq) to enable the high-throughput characterization of genetic parts controlling translation in absolute units. We simultaneously measure 743 translation initiation rates and 754 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 causes a σ32-mediated stress response, codon-specific pausing, and drop in translation initiation rates across the cell. Our work demonstrates the power of integrating global approaches to give a comprehensive and quantitative understanding of gene regulation and burden in living cells.
Read in full at bioRxiv.
This is an abstract of a preprint hosted on an independent third party site. It has not been peer reviewed but is currently under consideration at Nature Communications.