RT Journal Article
SR Electronic
T1 Absolute quantification of translational regulation and burden using combined sequencing approaches
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 338939
DO 10.1101/338939
A1 Thomas E. Gorochowski
A1 Irina Chelysheva
A1 Mette Eriksen
A1 Priyanka Nair
A1 Steen Pedersen
A1 Zoya Ignatova
YR 2018
UL http://biorxiv.org/content/early/2018/06/04/338939.abstract
AB 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.