title: RNA Structural Determinants of Optimal Codons Revealed by MAGE-Seq.
abstract: Synonymous codon choices at the beginning of genes optimize 5' RNA structures for enhanced translation initiation, but less is known about mechanisms that drive codon optimization downstream within the gene. To understand what determines codon choices across a gene, we generated 12,726 in situ codon mutants in the Escherichia coli essential gene infA and measured their fitness by combining multiplex automated genome engineering mutagenesis with amplicon deep sequencing (MAGE-seq). Correlating predicted 5' RNA structure with fitness revealed that codons even far from the start of the gene are deleterious if they disrupt the native 5' RNA conformation. These long-range structural interactions generate context-dependent rules that constrain codon choices beyond intrinsic codon preferences. Genome-wide RNA folding predictions confirm that natural codon choices far from the start codon are optimized in part to prevent disruption of native structures near the 5' UTR. Our results shed light on natural codon distributions and should improve engineering of gene expression for synthetic biology applications.
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA; Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Eric D
authors.initials: ED
authors.lastname: Kelsic
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Hattie
authors.initials: H
authors.lastname: Chung
authors.affiliation: Faculty of Physics, Technion - Israel Institute of Technology, Haifa 3200003, Israel.
authors.forename: Niv
authors.initials: N
authors.lastname: Cohen
authors.affiliation: Department of Systems Biology, Columbia University Medical Center, New York, NY 10032, USA.
authors.forename: Jimin
authors.initials: J
authors.lastname: Park
authors.affiliation: Department of Systems Biology, Columbia University Medical Center, New York, NY 10032, USA; Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY 10032, USA. Electronic address: hw2429@columbia.edu.
authors.forename: Harris H
authors.initials: HH
authors.lastname: Wang
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA; Faculty of Biology and Faculty of Computer Science, Technion - Israel Institute of Technology, Haifa 3200003, Israel. Electronic address: rkishony@technion.ac.il.
authors.forename: Roy
authors.initials: R
authors.lastname: Kishony
keywords: RNA structure; codon; codon optimization; codon usage; computational biology; molecular biology; synthetic biology; systems biology