Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 110, Issue 28, Pages 11284-11289Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1305590110
Keywords
eukaryotic translation; translation initiation site; Kozak consensus sequence; p21; synthetic biology
Categories
Funding
- National Science Foundation [0846392]
- Ellison Medical Foundation [AG-NS-0550-09]
- Div Of Chem, Bioeng, Env, & Transp Sys
- Directorate For Engineering [0846392] Funding Source: National Science Foundation
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We engineered short ORFs and used them to control the expression level of recombinant proteins. These short ORFs, encoding a two-amino acid peptide, were placed upstream of an ORF encoding a protein of interest. Insertion of these upstream ORFs (uORFs) resulted in suppression of protein expression. By varying the base sequence preceding the uORF, we sought to vary the translation initiation rate of the uORF and subsequently control the degree of this suppression. Using this strategy, we generated a library of RNA sequence elements that can specify protein expression over a broad range of levels. By also using multiple uORFs in series and non-AUG start codons, we were able to generate particularly low expression levels, allowing us to achieve expression levels spanning three orders of magnitude. Modeling supported a mechanism where uORFs shunt the flow of ribosomes away from the downstream protein-coding ORF. With a lower translation initiation rate at the uORF, more ribosomes leak past the uORF; consequently, more ribosomes are able to reach and translate the downstream ORF. We report expression control by engineering uORFs and translation initiation to be robust, predictable, and reproducible across all cell types tested. We propose control of translation initiation as a primary method of choice for tuning expression in mammalian systems.
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