title: Avoidance of truncated proteins from unintended ribosome binding sites within heterologous protein coding sequences.
abstract: Genetic sequences ported into non-native hosts for synthetic biology applications can gain unexpected properties. In this study, we explored sequences functioning as ribosome binding sites (RBSs) within protein coding DNA sequences (CDSs) that cause internal translation, resulting in truncated proteins. Genome-wide prediction of bacterial RBSs, based on biophysical calculations employed by the RBS calculator, suggests a selection against internal RBSs within CDSs in Escherichia coli, but not those in Saccharomyces cerevisiae. Based on these calculations, silent mutations aimed at removing internal RBSs can effectively reduce truncation products from internal translation. However, a solution for complete elimination of internal translation initiation is not always feasible due to constraints of available coding sequences. Fluorescence assays and Western blot analysis showed that in genes with internal RBSs, increasing the strength of the intended upstream RBS had little influence on the internal translation strength. Another strategy to minimize truncated products from an internal RBS is to increase the relative strength of the upstream RBS with a concomitant reduction in promoter strength to achieve the same protein expression level. Unfortunately, lower transcription levels result in increased noise at the single cell level due to stochasticity in gene expression. At the low expression regimes desired for many synthetic biology applications, this problem becomes particularly pronounced. We found that balancing promoter strengths and upstream RBS strengths to intermediate levels can achieve the target protein concentration while avoiding both excessive noise and truncated protein.
authors.affiliation: †Departments of Bioengineering, University of California, Berkeley, California 94720, United States.|§The University of California, Berkeley and University of California, San Francisco Graduate Program in Bioengineering, Berkeley, California 94720, United States.
authors.forename: Weston R
authors.initials: WR
authors.lastname: Whitaker
authors.affiliation: †Departments of Bioengineering, University of California, Berkeley, California 94720, United States.|‡Energy Biosciences Institute, University of California, Berkeley, 2151 Berkeley Way, Berkeley California 94704, United States.
authors.forename: Hanson
authors.initials: H
authors.lastname: Lee
authors.affiliation: †Departments of Bioengineering, University of California, Berkeley, California 94720, United States.|‡Energy Biosciences Institute, University of California, Berkeley, 2151 Berkeley Way, Berkeley California 94704, United States.|∥Physical Biosciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, California 94720, United States.
authors.forename: Adam P
authors.initials: AP
authors.lastname: Arkin
authors.affiliation: †Departments of Bioengineering, University of California, Berkeley, California 94720, United States.|‡Energy Biosciences Institute, University of California, Berkeley, 2151 Berkeley Way, Berkeley California 94704, United States.|∥Physical Biosciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, California 94720, United States.
authors.forename: John E
authors.initials: JE
authors.lastname: Dueber
mesh_terms: D001665:Binding Sites / Q000502:physiology*; D004926:Escherichia coli; D008958:Models, Molecular; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000502:physiology*; D012270:Ribosomes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D058615:Synthetic Biology
keywords: RBS calculator; gene optimization; internal ribosome binding sites; protein expression; truncated protein