# Five-prime cap
The five-prime cap (5′ cap) is an N7-methylated guanosine joined to the first nucleotide of a eukaryotic or viral messenger RNA through a reverse 5′-to-5′ triphosphate linkage, a structure found on essentially all eukaryotic mRNAs and essential for cell viability.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC5027499/) • [2](https://ncbi.nlm.nih.gov/pmc/articles/PMC2825737/) • [3](https://pmc.ncbi.nlm.nih.gov/articles/PMC3142331/) The cap was independently characterized in 1975 by [Bernard Moss](https://www.edgepedia.org/bernard-moss) with Cha-Mer Wei and by Aaron Shatkin with Yasuhiro Furuichi, who submitted their findings simultaneously.[4](https://pmc.ncbi.nlm.nih.gov/articles/PMC5354508/) It stabilizes the mRNA, directs splicing and nuclear export, and is required for efficient translation.[5](https://pmc.ncbi.nlm.nih.gov/articles/PMC3977584/)
| Key fact | Detail |
|---|---|
| Structure | m7G(5′)ppp(5′)X: an inverted 7-methylguanosine linked 5′-to-5′ through a triphosphate bridge to the first transcribed nucleotide[2](https://ncbi.nlm.nih.gov/pmc/articles/PMC2825737/) |
| Biosynthesis | Three enzymatic steps: RNA triphosphatase, guanylyltransferase via a lysyl-GMP intermediate, and N7 methyltransferase using SAM[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC5027499/) • [2](https://ncbi.nlm.nih.gov/pmc/articles/PMC2825737/) |
| Timing | Co-transcriptional, once the first 25–30 nucleotides of the nascent Pol II transcript exist[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC5027499/) |
| Cap variants | Cap 0 (m7GpppN), cap 1 (m7GpppNm), cap 2 (m7GpppNmpNm); cap 4 in trypanosomatids[6](https://pmc.ncbi.nlm.nih.gov/articles/PMC4729855/) • [7](https://pmc.ncbi.nlm.nih.gov/articles/PMC3962952/) |
| Decay | Dcp2/Nudt16 hydrolyze the cap to m7GDP, exposing a 5′ monophosphate for XRN1 5′→3′ exonucleolytic decay[8](https://pmc.ncbi.nlm.nih.gov/articles/PMC12860496/) |
| Immune marker | Cap 1 2′-O methylation contributes to self/non-self discrimination[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC5027499/) |
| Manufacturing | Enzymatic capping reaches 84–100% efficiency; ARCA analogs 34–77%; CleanCap 90–99%[9](https://pmc.ncbi.nlm.nih.gov/articles/PMC8879150/) • [8](https://pmc.ncbi.nlm.nih.gov/articles/PMC12860496/) |
## Chemical structure and cap variants
The cap is written m7G(5′)ppp(5′)X: the 7-methylguanosine is joined through its 5′ hydroxyl to the first transcribed nucleotide via a triphosphate linkage, the reverse orientation of the 3′-to-5′ phosphodiester bonds within the RNA body.[2](https://ncbi.nlm.nih.gov/pmc/articles/PMC2825737/) This inverted guanosine methylated at N7 is the defining part of the structure.[10](https://pubs.acs.org/doi/full/10.1021/acs.accounts.3c00442)
**Methylation states.** Cap 0 carries only the N7-methylguanosine; cap 1 adds 2′-O-methylation on the first transcribed nucleotide, catalyzed by CMTR1; cap 2 adds 2′-O-methylation on the second nucleotide, catalyzed by CMTR2.[8](https://pmc.ncbi.nlm.nih.gov/articles/PMC12860496/) Cap 0 and cap 1 methylation occur in the nucleus, whereas cap 2 modification occurs in the cytoplasm.[7](https://pmc.ncbi.nlm.nih.gov/articles/PMC3962952/) • [6](https://pmc.ncbi.nlm.nih.gov/articles/PMC4729855/) Organisms differ: yeasts contain mainly cap 0, higher organisms usually have more extensively methylated caps, and in trypanosomatids such as *Leishmania* and *Trypanosoma* the first four nucleosides are hypermethylated to generate the cap 4 structure through the methyltransferases TbMTr1, TbMTr2, and TbMTr3.[6](https://pmc.ncbi.nlm.nih.gov/articles/PMC4729855/) • [7](https://pmc.ncbi.nlm.nih.gov/articles/PMC3962952/)
**Alternative 5′ ends.** The m7G cap is not the only RNA 5′ modification. Others include 2,2,7-trimethylguanosine, 5′-γ-methyl phosphate, and dimethyl monophosphate caps, and RNAs capped with NAD or coenzyme A.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC5027499/) NAD-capped RNAs were first found in bacteria and yeast and later in mammalian cells, where they mark transcripts for degradation; NAD capping levels depend partly on cellular NAD concentration.[11](https://pmc.ncbi.nlm.nih.gov/articles/PMC6414751/) In *E. coli*, mass spectrometry in 2009 estimated about 3000 NAD-capped RNA copies per cell, enriched for regulatory small RNAs and 5′ mRNA fragments.[12](https://pmc.ncbi.nlm.nih.gov/articles/PMC4371356/)
## Biosynthesis and coupling to transcription
Capping proceeds in three steps. An RNA triphosphatase removes the terminal γ-phosphate from the transcription start nucleotide, leaving 5′ diphosphate RNA. A guanylyltransferase then reacts with the α-phosphate of GTP to form an enzyme–GMP intermediate (Gp-Enz) with release of pyrophosphate, and transfers the GMP to the RNA diphosphate end, producing GpppRNA. Finally, a guanine-N7 methyltransferase methylates the cap using S-adenosylmethionine as methyl donor, producing m7GpppRNA.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC5027499/) • [2](https://ncbi.nlm.nih.gov/pmc/articles/PMC2825737/) • [5](https://pmc.ncbi.nlm.nih.gov/articles/PMC3977584/) A 2′-O methyltransferase then converts cap 0 to cap 1.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC5027499/) The enzyme organization varies: in *Saccharomyces cerevisiae* three separate proteins (Cet1, Ceg1, and Abd1) carry the three activities, while in metazoans the triphosphatase and guanylyltransferase are fused in the bifunctional protein RNGTT.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC5027499/)
**Coupling to RNA polymerase II.** Capping is the first modification made to Pol II-transcribed RNA and occurs co-transcriptionally in the nucleus as soon as the first 25–30 nucleotides are incorporated.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC5027499/) The capping enzyme is recruited to the polymerase through the phosphorylated C-terminal domain (CTD) of Rpb1, which consists of tandem heptapeptide repeats with the consensus Y1S2P3T4S5P6S7. During initiation and early elongation the CTD is enriched in Ser5 and Ser7 phosphorylation; as elongation proceeds, Ser5 and Ser7 phosphorylation decrease while Ser2 phosphorylation increases.[7](https://pmc.ncbi.nlm.nih.gov/articles/PMC3962952/) Capping enzymes bind directly to the Ser5-phosphorylated CTD generated immediately after transcription initiation, a contact conserved among budding yeast, fission yeast, and mammals.[3](https://pmc.ncbi.nlm.nih.gov/articles/PMC3142331/) This coupling has a measurable payoff: co-transcriptional capping of Pol II transcripts proceeds orders of magnitude more efficiently than capping of free RNA.[13](https://www.nature.com/articles/s41467-018-05923-w)
## Functions in mRNA metabolism
The cap protects the mRNA 5′ end against 5′→3′ exonucleolytic degradation and increases the accuracy and efficiency of splicing; it is added during the initial phases of transcription, before internal N6-methyladenosine modification, 3′ poly(A) addition, and exon splicing.[6](https://pmc.ncbi.nlm.nih.gov/articles/PMC4729855/) A mature methylated cap is essential for recognition by the nuclear cap-binding complex (CBC) and by eIF4E, which coordinate splicing, nuclear export, translation, and stability.[5](https://pmc.ncbi.nlm.nih.gov/articles/PMC3977584/)
**Translation initiation.** In cap-dependent initiation, eIF4E binds the m7G cap at the mRNA 5′ end and associates with the 43S pre-initiation complex.[14](https://pmc.ncbi.nlm.nih.gov/articles/PMC7549709/) Newly exported transcripts first undergo a CBC-dependent pioneer round of translation, which precedes eIF4E-dependent steady-state translation after importin-β-mediated remodeling of the messenger ribonucleoprotein.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC5027499/)
## Decapping and mRNA decay
In 5′→3′ decay, the cap must first be removed. Decapping is catalyzed by the Nudix hydrolases Dcp2 (with its cofactor Dcp1) and Nudt16, which hydrolyze the triphosphate bridge between the α- and β-phosphates, releasing m7GDP and leaving a 5′-monophosphorylated mRNA that is a substrate for 5′→3′ exoribonucleases such as Xrn1/Rat1.[7](https://pmc.ncbi.nlm.nih.gov/articles/PMC3962952/) • [8](https://pmc.ncbi.nlm.nih.gov/articles/PMC12860496/) • [5](https://pmc.ncbi.nlm.nih.gov/articles/PMC3977584/) In mammalian cells, decay typically begins with deadenylation.[8](https://pmc.ncbi.nlm.nih.gov/articles/PMC12860496/) A separate quality-control layer exists: DXO family enzymes (Rai1 and Ydr370C/Dxo1 in yeast, Dom3Z/DXO in mammals) preferentially decap and degrade incompletely capped mRNAs, acting on unmethylated GpppN caps and converting them to substrates for XRN degradation, which links cap N7-methylation quality control to splicing and polyadenylation.[5](https://pmc.ncbi.nlm.nih.gov/articles/PMC3977584/) • [1](https://pmc.ncbi.nlm.nih.gov/articles/PMC5027499/)
## By the numbers
- **Capping window:** capping occurs once the first 25–30 nucleotides of the nascent transcript exist; the vaccinia capping enzyme acts on transcripts of at least 31 nucleotides.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC5027499/) • [7](https://pmc.ncbi.nlm.nih.gov/articles/PMC3962952/)
- **Capping efficiency in vitro:** enzymatically capped IVT mRNA measured 84–100% across 12 samples, while ARCA-capped samples measured 34–53% for hAg 5′UTR RNAs and 67–77% for TEV 5′UTR-containing RNAs.[9](https://pmc.ncbi.nlm.nih.gov/articles/PMC8879150/)
- **CleanCap:** co-transcriptional incorporation yields cap 1 mRNA at 90–99% capping efficiency.[8](https://pmc.ncbi.nlm.nih.gov/articles/PMC12860496/)
- **NAD caps:** about 3000 NAD-capped RNA copies per *E. coli* cell.[12](https://pmc.ncbi.nlm.nih.gov/articles/PMC4371356/)
- **Decapping-resistant cap:** CleanCap M6 increased RNA half-life approximately 2.5-fold in a reconstituted Dcp2/Xrn1 decay system, and up to 23-fold greater longevity of Fluc mRNA in HEK293T cells relative to AG 3′OMe-capped mRNA.[15](https://doi.org/10.1016/j.omtn.2025.102456)
- **Engineered polymerase:** T7-68 achieved a 4-fold reduction in required cap analog concentration and more than 50-fold reduced dsRNA byproduct generation versus wild-type T7 RNAP.[16](https://pubs.rsc.org/en/content/articlehtml/2024/fd/d4fd00023d)
## Viral caps and host–pathogen interplay
Viruses have evolved highly diverse capping mechanisms, using their own or cellular capping machineries, or stealing cap structures from cellular mRNAs.[17](https://www.nature.com/articles/nrmicro2675) [Influenza](https://www.edgepedia.org/influenza) virus uses cap snatching: the PB2 subunit binds cellular capped transcripts and the PA N-terminal endonuclease domain cleaves between nucleotides 10 and 13 to generate a short capped oligoribonucleotide primer for viral mRNA synthesis.[7](https://pmc.ncbi.nlm.nih.gov/articles/PMC3962952/) The vaccinia capping enzyme, a heterodimer of 97 kDa and 33 kDa subunits that catalyzes all three capping steps, was the tool through which the capping pathway was elucidated between 1975 and 1984.[12](https://pmc.ncbi.nlm.nih.gov/articles/PMC4371356/)
**Self versus non-self.** Cap 1 2′-O methylation of the +1 nucleotide contributes to innate immune discrimination of foreign RNA.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC5027499/) Unmethylated or incompletely capped RNA can trigger antiviral responses, which is why capping efficiency is a critical quality attribute in mRNA manufacture.[16](https://pubs.rsc.org/en/content/articlehtml/2024/fd/d4fd00023d)
## Biotechnological applications
**Enzymatic capping.** Capping with the vaccinia capping enzyme allows complete capping of the RNA, generating cap 0 RNA that can be converted to cap 1 with a cap-specific 2′-O methyltransferase.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC5027499/) Measured efficiencies across sequence contexts run 84–100%.[9](https://pmc.ncbi.nlm.nih.gov/articles/PMC8879150/)
**Co-transcriptional capping.** Cap analogs such as m7GpppG, ARCA, and CleanCap are incorporated during in vitro transcription with phage RNA polymerases. Co-transcriptional methods minimize the reaction steps and enzymes needed compared with multi-step enzymatic approaches and are more efficient than first-generation analogs such as ARCA.[18](https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cpz1.39) CleanCap incorporation yields cap 1 mRNA at up to 90–99% efficiency, and the Pfizer/BioNTech COVID-19 vaccine used the trinucleotide cap 1 analog m7GpppAmG.[8](https://pmc.ncbi.nlm.nih.gov/articles/PMC12860496/) Substituting modified nucleotides such as m5CTP and pseudouridine reduces TLR-mediated innate immune responses in the transcript body.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC5027499/) Cap analog choice also drives protein expression in a cell-line-dependent manner, as shown in the cap-analog library screening that developed the COVID-19 mRNA vaccine STP2104.[19](https://doi.org/10.3389/fimmu.2025.1571713) Liquid chromatography–tandem mass spectrometry methods now support analysis of mRNA identity, integrity, 5′ capping efficiency, and poly(A) tail length as critical quality attributes.[20](https://pmc.ncbi.nlm.nih.gov/articles/PMC11855904/)
## What has changed since 2023
**Cap epitranscriptomics.** [Attention](https://www.edgepedia.org/attention) has grown on cap-adjacent modifications beyond the canonical methylations: CAPAM methylates the N6 position of a cap-adjacent adenosine, and m6Am-related and epitranscriptome-inspired cap analogs, in which the first transcribed adenosine carries N6-methylation, have attracted increasing attention in vaccine cap engineering.[8](https://pmc.ncbi.nlm.nih.gov/articles/PMC12860496/)
**Manufacturing advances.** Several 2024–2025 results have changed the toolset. The engineered T7-68 RNA polymerase co-transcriptionally incorporates di- and trinucleotide cap analogs with high efficiency even at reduced cap analog concentrations, cutting required cap concentration 4-fold and dsRNA byproducts more than 50-fold.[16](https://pubs.rsc.org/en/content/articlehtml/2024/fd/d4fd00023d) A language-model-enabled screening platform has been used to discover new mRNA capping enzymes, motivated by the finding that incomplete capping reduces antigen production and can trigger adverse immune responses.[21](https://www.science.org/doi/10.1126/sciadv.adt0402) The decapping-resistant analog CleanCap M6 (m7G3′OMeppp m6A20OMepG) resists Dcp2-mediated decapping and correlated with substantially increased protein expression in vivo compared with mRNAs capped using existing industry standards.[15](https://doi.org/10.1016/j.omtn.2025.102456) A one-pot-two-step synthesis produced over 70 modified trinucleotide cap primers, analogs of 7mGpppAmpG; full complementarity of the primer to template positions +1 and +2 was required and sufficient to obtain capped mRNA with greater than 98% capping efficiency and greater than 5 mg/mL yield.[22](https://rnajournal.cshlp.org/content/early/2025/11/19/rna.080634.125)
## References
1. [mRNA capping: biological functions and applications, Nucleic Acids Research](https://pmc.ncbi.nlm.nih.gov/articles/PMC5027499/)
2. [Regulation of mRNA cap methylation](https://ncbi.nlm.nih.gov/pmc/articles/PMC2825737/)
3. [Structural insights to how mammalian capping enzyme reads the CTD code, PNAS](https://pmc.ncbi.nlm.nih.gov/articles/PMC3142331/)
4. [Investigating Viruses during the Transformation of Molecular Biology](https://pmc.ncbi.nlm.nih.gov/articles/PMC5354508/)
5. [Structure and Function of Pre-mRNA 5′-End Capping, Cold Spring Harbor Perspectives in Biology](https://pmc.ncbi.nlm.nih.gov/articles/PMC3977584/)
6. [Discovery of m7G-cap in eukaryotic mRNAs, Proceedings of the Japan Academy](https://pmc.ncbi.nlm.nih.gov/articles/PMC4729855/)
7. [Enzymology of RNA cap synthesis, WIREs RNA](https://pmc.ncbi.nlm.nih.gov/articles/PMC3962952/)
8. [The 5′ Cap Epitranscriptome and Beyond, PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC12860496/)
9. [Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA](https://pmc.ncbi.nlm.nih.gov/articles/PMC8879150/)
10. [Chemical Modifications of mRNA Ends for Therapeutic Applications, Accounts of Chemical Research](https://pubs.acs.org/doi/full/10.1021/acs.accounts.3c00442)
11. [mRNA cap regulation in mammalian cell function and fate, PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC6414751/)
12. [RNA capping: progress and prospects, RNA Biology](https://pmc.ncbi.nlm.nih.gov/articles/PMC4371356/)
13. [CTD-dependent and -independent mechanisms govern co-transcriptional capping of Pol II transcripts, Nature Communications](https://www.nature.com/articles/s41467-018-05923-w)
14. [The diversity, plasticity, and adaptability of cap-dependent translation initiation](https://pmc.ncbi.nlm.nih.gov/articles/PMC7549709/)
15. [CleanCap M6 inhibits decapping of exogenously delivered IVT mRNA, Molecular Therapy – Nucleic Acids](https://doi.org/10.1016/j.omtn.2025.102456)
16. [An engineered T7 RNA polymerase for efficient co-transcriptional capping with reduced dsRNA byproducts, Faraday Discussions](https://pubs.rsc.org/en/content/articlehtml/2024/fd/d4fd00023d)
17. [Conventional and unconventional mechanisms for capping viral mRNA, Nature Reviews Microbiology](https://www.nature.com/articles/nrmicro2675)
18. [Cap 1 Messenger RNA Synthesis with Co-transcriptional CleanCap Analog by In Vitro Transcription, Current Protocols](https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cpz1.39)
19. [Discovery and development of a safe and efficient COVID-19 mRNA vaccine, STP2104, Frontiers in Immunology](https://doi.org/10.3389/fimmu.2025.1571713)
20. [Characterisation and analysis of mRNA critical quality attributes using liquid chromatography based methods](https://pmc.ncbi.nlm.nih.gov/articles/PMC11855904/)
21. [Discovery of diverse and high-quality mRNA capping enzymes through a language model–enabled platform, Science Advances](https://www.science.org/doi/10.1126/sciadv.adt0402)
22. [Novel Trinucleotide mRNA Capping Reagents, RNA](https://rnajournal.cshlp.org/content/early/2025/11/19/rna.080634.125)
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*Topic: Encyclopedia › Life and health › Biological foundations › RNA and gene regulation › RNA processing, modification, and translation › mRNA end processing and export › 5′ capping*
*Initially written Sep 17, 2026 · Reviewed: Sep 30, 2026; Oct 21, 2026 · Edited: Sep 30, 2026; Oct 21, 2026 · Last review: Oct 21, 2026*
*Copyright 2026 EdgeChat AI, a subsidiary of Biostate AI.*
License: Edgepedia Community License 1.0, https://www.edgepedia.org/edgepedia/license