Guanosine triphosphate oligo capping 5 Gppp (2023)

5’ Guanosine-triphosphate Cap (5’ Gppp)

All newly formed eukaryotic RNA transcripts undergo a number of post-transcriptional modifications from the precursor 5' guanosine-triphosphate (5' Gppp) before they are transferred into the cytoplasm. Eukaryotic mRNAs are initially transcribed by RNA polymerase II as direct copies of a gene that include several non-coding sequences such as introns.

A nuclear RNA molecule experiences three main events during its life:

  1. The addition of a 5' cap,
  2. The addition of a 3' cap,
  3. The removal of unwanted intronic sequences by a process called RNA splicing.

The regulation of the intracellular deoxynucleotide triphosphate (dNTP) pool is important for genomic stability. It has been suggested that an imbalanced dNTP pool can lead to mutagenesis and cell proliferation. The result is the development of cancer.

The model of the mRNA capping enzyme in complex with the CAP analog GPPPG is shown in figure 1.

(Video) 5 Prime Capping of mRNA | Eukaryotic mRNA Processing

Guanosine triphosphate oligo capping 5 Gppp (1)

Figure 1: Structure of mRNA capping enzyme in complex with the CAP analog GPPPG from Paramecium bursaria Chlorella virus 1 [1CKO,Hâkansson K., and Wigley DB., 1998].

In eukaryotes, the mRNA 5' CAP structure is important for pre-mRNA processing, pre-mRNA splicing, mRNA export, translation initiation, and mRNA stability. Removal of the CAP targets the transcript for decay. Viruses use the CAP structure to ensure efficient translation of their RNA and to evade the host's immune sytem. However, structural and biophysical studies of capped RNA are difficult because the production of these compounds in large quantities are challenging.

Splicing is a particularly complex process and some eukaryotic cells can make different proteins by forming different mRNAs from the same gene, also called alternative splicing. In addition, the identification of post-translational modifications within mRNA molecules has been quite important in the development of in vivo therapeutic strategies, such as in anti-sense oligonuleotides based technologies, and in vitro gene manipulations, such as in the construction of cDNA libraries.

During transcription eukaryotic RNA becomes associated with small ribonuclear proteins that add a cap structure such as guanosine triphosphate (5' Gppp) to the 5’ end. The majority of eukaryotic and viral messenger RNAs apparently contain a cap structure. RNA molecules that are 20 to 30 nucleotides long are capped by the initial addition of a guanine nucleotide to the first RNA base by a reaction catalyzed by the enzyme guanalyl transferase.

Transcription starts with a nucleotide triphosphate which is usually a purine guanosine triphosphate or adenosine triphoshate. The first nucleotide has a 5’ triphosphate group and the usual phosphodiester bond on its 3’ position which is connected to the 5’ position of the next nucleotide. The initial sequence is: 5’ guanosine-triphosphate (Gppp) or 5' adenosine-triphosphate (Appp) pNpNpNp… . The 5’ 7-methylguanosine (m7G) cap is a unique feature of eukaryotic cellular and viral mRNA and small nuclear and nucleolar RNAs involved in nuclear RNA processing reactions. Capping appears to be essential for eukaryotic cells. The cap is attached backwards by the formation of a 5’ to 5’ linkage of the first RNA base. The N7 atom of the guanine is also methylated following the addition. This reaction generates a 7-methyl guanine and this cap structure is also called a “cap 0” structure. Furthermore, the adjacent ribose units can also be methylated at the C2 position. If the C2 position at the first ribose is methylated the cap is called “cap 1” structure, and if the C2 positions on both riboses are methylated it is called a “cap 2” structure.

(Video) RNA Processing | 5' Capping & Polyadenylation | Types of Intron | Intron Splicing | CSIR NET 2020

The "cap O" structure, N7-methylguanosine-triphosphate (7mGpppN), at the 5′-terminus is required in varying degrees for processing and maturation of the RNA transcript in the nucleus. The precursor to the N7-MeGppp cap is the unmethylated version, 5′ guanosine-triphosphate (Gppp). The cap is formed by three enzymatic reactions at the 5’ terminus of the nascent mRNAs.

Structures of the different m7G caps and of the precursor of the N7-MeGppp cap which is the unmethylated version, 5′ guanosine-triphosphate (Gppp), are shown in table 1.

Table 1: Cap Structures.

Guanosine triphosphate oligo capping 5 Gppp (2) Guanosine triphosphate oligo capping 5 Gppp (3)
A) m7G cap cap O” structure (7-methyl guanine) B)"cap 1" structure; 2' mono methylated
Guanosine triphosphate oligo capping 5 Gppp (4) Guanosine triphosphate oligo capping 5 Gppp (5)
C) “cap 2” structure; 2’ dimethylated D) Precursor for the m7G cap

Functions of CAPs

CAPs are believed to serve several functions. They may be

(Video) Chemical synthesis of oligonucleotides|Phosphoramidite Method|

(i) involved in subsequent splicing reactions,

(ii) and/or serve as positioning guides for translation.

The observation that ribosomes can not bind to uncapped messages supports this notion. Also, capping may also determine the stability of transcripts explaining observed differences in the half-lives between prokaryotic and eukaryotic mRNAs. In addition, the observation that CAP analogs can inhibit splicing when added at the start of the reaction but not at later times of incubation suggests that the CAP recognition is an important step in the formation of a specific ribonucleoprotein complex required for splicing. Several studies including X-ray crystallography have now shown that the cap structure is necessary for optimal mRNA translation, that it participates in the splicing of mRNA precursors, and affects nuclear export and mRNA stability. Furthermore, the CAP's function in translation is mediated by the 25 kDa protein eukaryotic initiation factor 4E (eIF4E).

Different forms of CAPs, for example, m7GpppN on mRNAs and m2'7GpppN on low-molecular-weight nuclear RNAs, may interact with proteins that may be needed for the generation of mature cellular mRNAs. Studies using affinity photolabeling methods indicated that the cap binding activity in influenza virus is associated with a P protein.Figure 2 shows a summary of the possible different modifications on the 5’ guanosine-triphosphate CAP.

Guanosine triphosphate oligo capping 5 Gppp (6)

(Video) Easy ISI 5. Genetic Vaccine platforms mRNA

Figure 2: 5' Structure(s) and Functions of Guanosine-triphosphate CAPs.
The CAP blocks the 5' end of mRNA and may be methylated at several positions.

Synthesis and purification of 5' Guanosine Triphosphate Cap Oligonucleotides
- 5'Gppp -

5' Guanosine triphosphate cap RNA oligonucleotides can be synthesized enzymatically by in vitro transcription and chemically using phosphoramidite chemistries. The use of chemical synthesis allows reproducibly obtaining 5' guanosine triphosphate capped RNAs (5'Gppp) at almost any scale independent from the RNA sequences and allows the addition of modifications at specific positions as well. The resulting modified oligonucleotides can be purified to a high purity and analyzed by a number of established methods, including PAGE, reverse phase HPLC, anion-exchange HPLC and mass-spectrometry. Since free 2’, 3’-hydroxyls can form complexes with borate ions, caps or capped oligonucleotides can be purified by affinity chromatography using substrates such as dihydroxyboryl-cellulose. Treatment with periodate allows to oxidize the cis-diols to dialdehyde which can then be reduced with 3H borohydrate to label the terminus radioactively. Alternatively, fluorophores may be added using this chemistry as well.

Bio-Synthesis also offers synthetic capped RNA oligonucleotides that contain the N7-methylguanosine instead of unmodified guanosine in

µg to mg quantities.Please visit: 5' Cap oligonucleotide synthesis.


Hâkansson K, Wigley DB.; Structure of a complex between a cap analogue and mRNA guanylyl transferase demonstrates the structural chemistry of RNA capping. Proc Natl Acad Sci U S A. 1998 Feb 17;95(4):1505-10.

Hakansson K, Doherty AJ, Shuman S, Wigley DB; X-ray crystallography reveals a large conformational change during guanyl transfer by mrna capping enzymes.
Cell (Cambridge,Mass.) (1997) 89 p.545.

Kozak M. Structural features in eukaryotic mRNAs that modulate the initiation of translation.
J Biol Chem. 1991 Oct 25;266(30):19867-70.

HANS KROATH AND AARON J. SHATKIN; mRNA 5'-Cap Binding Activity in Purified Influenza Virus. JOURNAL OF VIROLOGY, Mar. 1982, p. 1105-1108 Vol. 41, No. 3.

Lewin, Benjamin; Genes IV. Oxford University Press 1990.

Laura O'Mullane and Ian C. Eperon; The pre-mRNA 5' cap determines wether U6 small nuclear RNA succeeds U1 small nucear ribonculeoprotein particle at 5' splice sites.
Mol. Cell. Biol. 1998, 18(12):7510-20.

Anna Niedzwiecka, Joseph Marcotrigiano, Janusz Stepinski, Marzena Jankowska-Anyszka, Aleksandra Wyslouch-Cieszynska, Michal Dadlez, Anne-Claude Gingras, Pawel Mak, Edward Darzynkiewicz, Nahum Sonenberg, Stephen K. Burley and Ryszard Stolarski; Biophysical Studies of eIF4E Cap-binding Protein: Recognition of mRNA 50 Cap Structure and Synthetic Fragments of eIF4G and 4E-BP1 Proteins.
J. Mol. Biol. (2002) 319, 615–635.

Shatkin AJ. Capping of eucaryotic mRNAs.
Cell. 1976 Dec;9(4 PT 2):645-53.

(Video) Capping of Eukaryotic mRNA



What does the 5 capping enzyme do? ›

5' capping is essential for mRNA stability, enhancing mRNA processing, mRNA export and translation. After successful capping, an additional phosphorylation event initiates the recruitment of machinery necessary for RNA splicing, a process by which introns are removed to produce a mature mRNA.

What is GPPP in mRNA? ›

The precursor to the N7-MeGppp cap is the unmethylated version, 5′ guanosine-triphosphate (Gppp). The cap is formed by three enzymatic reactions at the 5' terminus of the nascent mRNAs.

What is 5 guanosine triphosphate cap? ›

In eukaryotes, the 5′ cap (cap-0), found on the 5′ end of an mRNA molecule, consists of a guanine nucleotide connected to mRNA via an unusual 5′ to 5′ triphosphate linkage. This guanosine is methylated on the 7 position directly after capping in vivo by a methyltransferase.

What is the function of the 5 cap and poly A tail? ›

5' cap and poly-A tail

Both the cap and the tail protect the transcript and help it get exported from the nucleus and translated on the ribosomes (protein-making "machines") found in the cytosol 1start superscript, 1, end superscript.

What protein binds 5 cap? ›

A key participant in regulation of translation is eIF4E, the mRNA 5' cap-binding protein.

What does 5 cap mean? ›

If that same investor paid $20 million for the same property, but still only earned $1 million in net operating income, we'd refer to this as a 5-cap. This is the same concept as an investor who purchases an entire company for $20 million.

How is 5 cap added to mRNA? ›

5' End Capping

The cap is added by the enzyme guanyl transferase. This enzyme catalyzes the reaction between the 5' end of the RNA transcript and a guanine triphosphate (GTP) molecule. The figure above simply illustrates the reaction between the 5' end of the RNA transcript and the GTP molecule.

What is 5 capping in RNA processing? ›

5′-end capping occurs very early during Pol II transcription, typically after the synthesis of ∼20 nucleotides of the pre-mRNA. Capping has been linked to splicing and 3′-end processing of the pre-mRNA, and the export of the mature mRNA.

Why is capping important in mRNA? ›

The mRNA cap is a highly methylated modification of the 5′ end of RNA pol II-transcribed RNA. It protects RNA from degradation, recruits complexes involved in RNA processing, export and translation initiation, and marks cellular mRNA as “self” to avoid recognition by the innate immune system.

Why is guanosine triphosphate GTP important in the body? ›

It is used as a source of energy for protein synthesis and gluconeogenesis. GTP is essential to signal transduction, in particular with G-proteins, in second-messenger mechanisms where it is converted to guanosine diphosphate (GDP) through the action of GTPases.

What does guanosine triphosphate GTP do? ›

The function of GTP is to induce a conformational change in a macromolecule by binding to it. Since it is easily hydrolyzed by various GTPases, the use of GTP as a controlling element allows cyclic variation in macromolecular shape.

What is 5 cap made of? ›

The 5′ cap consists of 7-methylguanosine (m7G) linked by a 5′–5′-triphosphate bridge to messenger RNA (mRNA) and acts as the master regulator of mRNA turnover and translation initiation in eukaryotes.

What is the importance of 5 prime capping? ›

The 5' cap structure m7GpppN (where N is any nucleotide) is a ubiquitous feature of cellular eukaryotic mRNAs. The cap is multifunctional as it is involved in translation, nucleocytoplasmic transport, splicing, and stabilization of mRNA against 5' exonucleolytic degradation.

What is the importance of 5 end capping and polyadenylation of mRNA? ›

Capping has been linked to splicing and 3′-end processing of the pre-mRNA, and the export of the mature mRNA. In addition, the 5′-end cap is directly recognized by the eukaryotic translation initiation factor eIF-4E, which is essential for mRNA translation by the ribosome.

What is the purpose of the poly A tail? ›

The poly-A tail makes the RNA molecule more stable and prevents its degradation. Additionally, the poly-A tail allows the mature messenger RNA molecule to be exported from the nucleus and translated into a protein by ribosomes in the cytoplasm.

How does poly A tail prevent degradation? ›

Poly(A) tails provide toeholds where polynucleotide phosphorylase can initiate exonucleolytic degradation of tightly folded RNAs protected from exoribonucleases by 3' stable secondary structures.

What is the purpose of CAP binding protein? ›

Molecular Basis of Memory

eIF4E, the cap-binding protein promotes protein synthesis by binding to eIF4G. Beside its role in translation regulation, eIF4E is also important for mRNA stability and the proper subcellular localization of mRNA.

Is a 5 cap good? ›

Market analysts say an ideal cap rate is between five and 10 percent; the exact number will depend on the property type and location. In comparison, a cap rate lower than five percent denotes lesser risk but a more extended period to recover an investment.

What are the two main roles of the 5 cap of a eukaryotic mRNA? ›

The 5′-7-methylguanosine cap on eukaryotic mRNAs serves both to stimulate canonical translation initiation and block an alternative pathway.

Why is high cap rate high risk? ›

It indicates that a lower value cap rate corresponds to better valuation and a better prospect of returns with a lower level of risk. On the other hand, a higher value of cap rate implies relatively lower prospects of return on property investment, and hence a higher level of risk.

What is the 5 end of mRNA capped by? ›

The cap consists of a guanine nucleotide methylated at the N7 position, and is linked to the 5′ nucleotide of the RNA through an unusual 5′-5′ pyrophosphate linkage (m7GpppN) (Shatkin, 1976; Furuichi and Shatkin, 2000).

Is 5 capping the first step in mRNA processing? ›

Capping. As might be expected, the addition of an mRNA cap at the 5' end is the first step in mRNA processing, since the 5'end of the RNA is the first to be made. Capping occurs once the first 20-30 nucleotides of the RNA have been synthesized.

What would happen to mRNA that doesn't have a 5 cap? ›

What would happen to an mRNA that doesn't have a 5' cap? The finished mRNA molecule would not be able to dissociate from the polymerase. The polymerase would not recognize the mRNA and thus would not bind.

Where does mRNA get capped? ›

mRNA capping in eukaryotes

Capping is the first modification made to RNA polymerase II-transcribed RNA and takes place co-transcriptionally in the nucleus as soon as the first 25–30 nts are incorporated into the nascent transcript (6,7).

What is capping and why is it necessary? ›

Capping is an essential step for stability of nascently transcribed RNA and complete its life cycle successfully [35]. Otherwise, RNA is degraded by exonucleases. Capping is also necessary for further processing, splicing, localization and reading of RNAs. Process of capping begins alongwith transcription itself.

Is all mRNA capped? ›

All eukaryotic mRNA contains a cap structure - an N7-methylated guanosine linked to the first nucleotide of the RNA via a reverse 5′ to 5′ triphosphate linkage (Figure 1).

Does GTP activate G protein? ›

G proteins are molecular switches that are activated by receptor-catalyzed GTP for GDP exchange on the G protein alpha subunit, which is the rate-limiting step in the activation of all downstream signaling.

How does GTP regulate protein function? ›

Binding of GTP inherently changes the activity of the G proteins and increases their activity, through the loss of inhibitory subunits. In this more active state, G proteins can bind other proteins and turn on downstream signalling targets.

What is the significance of GTP in protein synthesis? ›

Protein synthesis rate as a function of [GTP]. Although the ATP concentration is probably the single most important parameter influencing the entire protein synthesis system, GTP is required for translation initiation, for elongation, and for termination.

Where is GTP produced? ›

In many organisms, succinyl-CoA synthetase of the citric acid cycle produces GTP from GDP in mitochondria.

What is difference between ATP and GTP? ›

ATP is utilized as a general cellular energy carrier4 and a phosphoryl donor in phosphorylation5. For this broad role of ATP, various enzymes bind ATP for their functions in the cell. GTP, on the other hand, is found to have a narrower range of cellular functions in, for instance, protein synthesis6 and signaling7.

How many GTP and ATP are required to synthesize a protein? ›

So, 1 ATP and 4 GTP molecules are used for each single amino acid incorporated into the peptide chain.

What has been removed from the pre-mRNA to make it into mRNA? ›

What has been removed from the pre-mRNA to make it into mRNA? Introns.

What means capping? ›

Well, to keep it simple, capping means "to lie." This slang term comes from the root slang word cap, which is "a lie." However, it shouldn't be confused with the other slang meaning of cap, which is "a bullet." Capping isn't a new word to TikTok or even Twitter for that matter.

What is the importance of capping in translation? ›

Capping protects mRNAs at their termini against attack by phosphatases and other nucleases and promotes mRNA function at the level of initiation of translation.

What is the purpose of capping the 5 end of mRNA quizlet? ›

What is the function of capping? The cap protects the 5' end of the mRNA from degradation by nucleases and also helps to position the mRNA correctly on the ribosomes during protein synthesis.

What is 5 capping in post transcriptional modification? ›

The 5' capping reaction replaces the triphosphate group at the 5' end of the RNA chain with a special nucleotide that is referred to as the 5' cap. It is thought to help with mRNA recognition by the ribosome during translation. A modification also takes place at the opposite end of the RNA transcript.

Why is capping important in RNA processing? ›

In the eukaryotic cell, capping of mRNA 5′ ends is an essential structural modification that allows efficient mRNA translation, directs pre-mRNA splicing and mRNA export from the nucleus, limits mRNA degradation by cellular 5′–3′ exonucleases and allows recognition of foreign RNAs (including viral transcripts) as 'non- ...

What does capping do to RNA? ›

RNA Capping

The mRNA cap structure engages critical translation factors to recruit ribosomes to mRNAs, promoting translation. Cap structures can be added to in vitro transcripts in two ways: After transcription by using capping enzymes, GTP and S-adenosyl methionine (SAM) During transcription by including cap analogs.

What are the benefits of capping? ›

First, consistency in capping helps to avoid waste, including the loss of product. An unsealed or loose cap on a product probably means that the bottle, cap and product will all be lost. Crooked or loose caps can lead to spills either on the production line or, even worse, once the product reaches the shelf.

How do you explain capping? ›

Capping is the act of capturing imagery of videos of others performing sexual acts without their knowledge or consent. WePROTECT also include in the definition of capping the act of capturing innocuous imagery of children and using it for sexual purposes.

Where does RNA capping occur? ›

RNA capping is required for both translation initiation as well as to dampen the host immune response to uncapped RNA. The host cell RNA capping machinery is restricted to the nucleus, while viral replication, transcription, and capping must occur in the ROs and/or in the cytoplasm.

Why is the 5 methyl G cap useful for the mRNA transcript? ›

This 5′ cap is a characteristic of nearly every mRNA molecule (17). Many functions have been ascribed to the cap, the most notable of which is protection of the mRNA from degradation by exonucleases. The cap may also promote splicing and nuclear export of the RNA and is recognized by the translational machinery.

What protects the 5 end of mRNA? ›

A 7-methylguanosine cap is added to the 5′ end of the pre-mRNA while elongation is still in progress. The 5′ cap protects the nascent mRNA from degradation and assists in ribosome binding during translation.

How does the 5 cap initiate translation? ›

The cap complex initiates protein translation by binding to the 5' cap of an mRNA and recruiting ribosomes to begin translation. Compared to animals and yeast, there are significant plant-specific differences in the regulation of cap-dependent mRNA translation, but these are poorly understood.

Why is the 5 cap and the poly A tail added to mRNA? ›

The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency.

What recognizes the 5 cap of mRNA transcripts? ›

In addition, the 5′-end cap is directly recognized by the eukaryotic translation initiation factor eIF-4E, which is essential for mRNA translation by the ribosome.


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