Poster abstracts
Poster number 23 submitted by Christopher Bonar
Synonymous codon substitutions enhance transcription and translation of an upstream gene in E. coli
Christopher D. Bonar (University of Notre Dame), Jacob D. Diehl (University of Notre Dame), Anabel Rodriguez (University of Notre Dame), Patricia L. Clark (University of Notre Dame)
Abstract:
Synonymous codon substitutions are genetic mutations that do not change the encoded protein sequence. Although often assumed to be phenotypically silent, synonymous codon substitutions can cause diverse effects on mRNA transcription and protein translation. Yet, the full range of effects of synonymous codon substitutions on the production and maintenance of a functional proteome remains poorly understood. We are using the well characterized Tet ON/Tet OFF divergent expression system to broadly test the effects of synonymous codon substitutions on gene expression. In Tet ON/Tet OFF, the expression level of TetR, encoded upstream of a gene of interest (GOI), regulates expression of both the GOI and tetR, by binding to and repressing a divergent promoter between the GOI and tetR. We found that synonymous mutations within a GOI can enhance transcription from a transcriptional start site (TSS) identified within the GOI. The effects of specific codon substitutions on transcription from this intragenic TSS are poorly predicted by existing prediction algorithms. Surprisingly, we found that transcription from this TSS bypasses canonical repression of tetR transcription. As a result, TetR protein accumulation increases, repressing GOI expression. This mechanism of transcription enhancement was corroborated by inserting an antisense terminator downstream of the intragenic TSS, which led to the expected reduction in tetR mRNA level. Even more surprisingly, although the mRNA produced from the intragenic TSS constitutes a minor fraction of tetR-encoding RNA, its level is predictive of TetR abundance, whereas total tetR mRNA level is not. We are currently testing whether this unexpected correlation between TetR and the minor fraction of tetR mRNA originating from the intragenic TSS is due to enhanced translational efficiency of the longer mRNA, or whether the longer RNA serves as a regulator of translational efficiency of canonical tetR mRNA. Collectively, these results demonstrate that synonymous codon substitutions can enhance intragenic RNA transcription to regulate upstream gene expression.
References:
Rodriguez, A., Diehl, J.D., Wright, G.S., Bonar, C.D., Lundgren, T.J., Moss, M.J., Li, J., Milenkovic, T., Huber, P.W., Champion, M.M., Emrich, S.J., and Clark, P.L. (2024). Synonymous codon substitutions modulate transcription and translation of a divergent upstream gene by modulating antisense RNA production. Proc. Natl. Acad. Sci. U. S. A. 121, e2405510121, 1–10.
Keywords: Synonymous codon substitutions, Intragenic transcriptional start site, Gene expression