Insertion mutagenesis to increase secondary structure within the 5′ noncoding region of a eukaryotic mRNA reduces translational efficiency
Abstract
The thymidine kinase gene of herpes simplex virus 1 was mutated by inserting oligodeoxynucleotide linkers into the region of the gene corresponding to the 5' untranslated portion of the mRNA. These linkers, when transcribed into mRNA, might be expected to form hairpin loops and hence to increase the secondary structure of the 5' end of the mRNA. Thymidine kinase insertion derivatives were examined in vivo and in vitro to determine translational efficiency. For the in vivo studies, thymidine kinase insertion derivatives were transfected into thymidine kinase deficient L cells alone and together with a selectable dominant marker, or were assayed in the COS-1 transient expression system. For in vitro studies, thymidine kinase insertion derivatives were subcloned into pSP64. Capped transcripts were analyzed for their ability to bind ribosomes and translate in rabbit reticulocyte lysates and wheat-germ extracts. The results demonstrate that translation efficiency is decreased as the number of linkers is increased and support the view that excessive secondary structure at the 5' end of eukaryotic mRNA impedes translation.
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Stable secondary structure in the 5′-UTR before or flanking the start codon decreases protein production.
“As demonstrated in previous studies on translation initiation in yeast [55] and in mouse cell lines [56], stable secondary structure in the 5′-UTR before or flanking the start codon decreases protein production.”
From Detailed Dissection and Critical Evaluation of the Pfizer/BioNTech and Moderna mRNA Vaccines (2021) · cites this paper as [56]
Higher GC content and highly negative folding minimal free energy (ΔG) of 5′UTRs correlate with inhibition of translation.
“In addition to greater length, the TPL sequence has a higher GC content and highly negative folding minimal free energy (ΔG), which correlates with inhibition of translation [4,7,45,46] (Table 1).”
From Effects of Combinations of Untranslated-Region Sequences on Translation of mRNA (2023) · cites this paper as [45]
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Effects of Combinations of Untranslated-Region Sequences on Translation of mRNA
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- Authors
- Pelletier, J., & Sonenberg, N.
- Affiliations
- McGill University (CA)
- Venue
- Cell
- Publisher
- Elsevier BV
- Published
- 1985-03
- Volume
- 40
- Issue
- 3
- Pages
- 515-526
- ISSN
- 0092-8674 (ISSN-L)
- OpenAlex
- W2021978074
- Cited by
- 610
- References
- 0
- Citations by year
- 2026: 10 · 2025: 9 · 2024: 12 · 2023: 12 · 2022: 12 · 2021: 8 · 2020: 10 · 2019: 9 · 2018: 7 · 2017: 9 · 2016: 10 · 2015: 8 · 2014: 11 · 2013: 4 · 2012: 9
- FWCI
- 20.18
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Cell (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Medical Research Council Canada
- National Cancer Institute
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Keywords
- insertional mutagenesis, 5' untranslated region, mRNA secondary structure, thymidine kinase gene, herpes simplex virus type 1, stem-loop structure, translation efficiency, ribosome binding, eukaryotic mRNA, dominant selectable marker
- MeSH terms
- Animals, Base Sequence, Cattle, Cell Line, Cloning, Molecular, DNA, Recombinant, DNA, Viral, Drug Resistance, Mutation, Neomycin, Nucleic Acid Conformation, Plasmids, Ribosomes, RNA, Messenger, Thymidine Kinase, Transfection, Protein Biosynthesis, Simplexvirus, Mice
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