Influences of mRNA secondary structure on initiation by eukaryotic ribosomes.
Open accessAbstract
Oligonucleotides designed to create hairpin structures were inserted upstream from the ATG initiator codon in several plasmids that encode preproinsulin, and the effects on translation were monitored in COS cells transfected by the vectors. Creation of a hairpin (delta G = -30 kcal/mol) that directly involves the ATG triplet at the start of the preproinsulin coding sequence does not reduce the yield of proinsulin. However, a more stable stem-and-loop structure (delta G = -50 kcal/mol) reduces the proinsulin yield by 85-95%. The stable hairpin inhibits even when it occurs at the midpoint of the 5' untranslated sequence and thus involves neither the cap nor the ATG codon. Presumably the migrating 40S ribosomal subunit can melt moderately stable duplexes but stalls at structures (delta G = -50 kcal/mol) that resist unfolding. Other experiments argue against the idea that sequestering the 5'-proximal ATG codon in a hairpin structure might allow it to be skipped by ribosomes in favor of an exposed ATG triplet farther downstream: when the primary sequence around the first ATG triplet is favorable for initiation, no translation from a downstream site can be detected, irrespective of whether the first ATG codon is single-stranded or base-paired.
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The paper's data captured and quantitated the repressive effect of secondary structure on ribosome load.
“By calculating UTR minimum free energies (MFE)26 and comparing them to UTR MRLs, we captured and quantitated this repressive effect of secondary structure on ribosome load (Supplementary Fig. 2c)17,25.”
From Human 5′ UTR design and variant effect prediction from a massively parallel translation assay (2019) · cites this paper as [25]
The paper reports that highly stable 5′-UTR hairpins inhibit translation by 85–95%, supporting a repressive effect of secondary structure under those conditions. It does not report quantifying ribosome load; the Discussion instead distinguishes inhibition of translation from initial 40S-subunit binding. The citing authors’ calculation of MFE and comparison with ribosome load are their own analysis, not a measurement in this source. (LLM, conf=0.84)
Experimental — accuracy not yet measuredEfficient translation initiation in mammalian species depends mainly on the secondary structure that may embed the Kozak consensus to obscure essential translation initiation signals.
“Efficient translation initiation in mammalian species depends mainly on two factors [40,41]: (1) the Kozak consensus [42,43,44], and (2) the secondary structure that may embed the Kozak consensus to obscure these essential translation initiation signals [9,41,45].”
From Detailed Dissection and Critical Evaluation of the Pfizer/BioNTech and Moderna mRNA Vaccines (2021) · cites this paper as [45]
The study description reports that a very stable hairpin inhibits translation, supporting a role for secondary structure. But the Discussion says moderately stable hairpins often have no effect and argues against the proposed mechanism of hiding an upstream start codon. It also identifies the cap and sequences flanking the ATG codon as other regulators, so this paper does not establish that initiation depends mainly on the two stated factors or that secondary structure inhibits it by obscuring the Kozak consensus. (LLM, conf=0.88)
Experimental — accuracy not yet measuredTranslation initiation and elongation is affected by RNA secondary structure that forms within 5′ UTRs and coding sequences, with strong structures showing the most negative effect on translation.
“Translation initiation and elongation is also affected by RNA secondary structure that forms within 5′ UTRs and coding sequences, with strong structures showing the most negative effect on translation17,25.”
From Human 5′ UTR design and variant effect prediction from a massively parallel translation assay (2019) · cites this paper as [25]
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Research works printed but not linked: 3
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- [19]Nomoto, A., Omata, T., Toyoda, H., Kuge, S., Horie, H., Kataoka, Y., Genba, Y., Nakano, Y. & Imura, N. (1982) Proc. Natl. Acad. Sci. USA 79, 5793–5797.
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- Authors
- Kozak, M.
- Affiliations
- University of Pittsburgh (US)
- Corresponding author
- Kozak, M
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- National Academy of Sciences
- Published
- 1986-05
- Volume
- 83
- Issue
- 9
- Pages
- 2850-2854
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC323404
- OpenAlex
- W2072971665
- Cited by
- 687
- References
- 0
- Citations by year
- 2026: 18 · 2025: 14 · 2024: 11 · 2023: 13 · 2022: 15 · 2021: 20 · 2020: 7 · 2019: 15 · 2018: 10 · 2017: 11 · 2016: 6 · 2015: 14 · 2014: 14 · 2013: 11 · 2012: 12
- FWCI
- 14.66
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institute of General Medical Sciences (GM 33915)
- 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 › RNA modifications and cancer
- Keywords
- mRNA secondary structure, translation initiation, ribosome scanning, hairpin structure, stem-loop structure, preproinsulin, 5' untranslated region, 40S ribosomal subunit, ribosome stalling
- MeSH terms
- Base Composition, Codon, Insulin, Mutation, Nucleic Acid Conformation, Proinsulin, Protein Precursors, Ribosomes, RNA, Messenger, Protein Biosynthesis
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