High-resolution profiling reveals coupled transcriptional and translational regulation of transgenes
Name
gkaf528.pdf
Description
Published version
Size
1.81 MB
Format
Adobe PDF
Checksum (MD5)
6a0037812a2c76efe5583a22a29d3c99
Author(s)
Peterman, Emma L
Ploessl, Deon S
Love, Kasey S
Sanabria, Valeria
Daniels, Rachel F
Johnstone, Christopher P
Godavarti, Diya R
Kabaria, Sneha R
Oakes, Conrad G
Pai, Athma A
Date Issued
June 6, 2025
Journal
Nucleic Acids Research
Publisher
Oxford University Press
Citation
Emma L Peterman, Deon S Ploessl, Kasey S Love, Valeria Sanabria, Rachel F Daniels, Christopher P Johnstone, Diya R Godavarti, Sneha R Kabaria, Conrad G Oakes, Athma A Pai, Kate E Galloway, High-resolution profiling reveals coupled transcriptional and translational regulation of transgenes, Nucleic Acids Research, Volume 53, Issue 11, 24 June 2025.
Version
Final published version
Abstract
Concentrations of RNAs and proteins provide important determinants of cell fate. Robust gene circuit design requires an understanding of how the combined actions of individual genetic components influence both messenger RNA (mRNA) and protein levels. Here, we simultaneously measure mRNA and protein levels in single cells using hybridization chain reaction Flow-FISH (HCR Flow-FISH) for a set of commonly used synthetic promoters. We find that promoters generate differences in both the mRNA abundance and the effective translation rate of these transcripts. Stronger promoters not only transcribe more RNA but also show higher effective translation rates. While the strength of the promoter is largely preserved upon genome integration with identical elements, the choice of polyadenylation signal and coding sequence can generate large differences in the profiles of the mRNAs and proteins. We used long-read direct RNA sequencing to define the transcription start and splice sites of common synthetic promoters and independently vary the defined promoter and 5′ UTR sequences in HCR Flow-FISH. Together, our high-resolution profiling of transgenic mRNAs and proteins offers insight into the impact of common synthetic genetic components on transcriptional and translational mechanisms. By developing a novel framework for quantifying expression profiles of transgenes, we have established a system for building more robust transgenic systems.
MIT Department
Massachusetts Institute of Technology. Department of Chemical Engineering
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Creative Commons Attribution-Noncommercial
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DOI of Published Version
https://doi.org/10.1093/nar/gkaf528
