The 5'-ends of all eukaryotic pre-mRNAs studied thus far are converted to cap structures. The cap is thought to influence splicing of the first intron, and is bound by 'cap-binding' proteins, CBP80 and CBP20, in the nucleus. The cap is important for translation initiation, and it also interacts with the poly(A)terminus, via proteins, resulting in circularization of the mRNA to facilitate multiple rounds of translation. The cap is also important for mRNA stability, protecting it from 5' to 3' nucleases, and is required for mRNA export to the cytoplasm. The capping reaction usually occurs very rapidly on nascent transcripts; after the synthesis of only a few nucleotides by RNA polymerase II. The capping reaction involves the conversion of the 5'-end of the nascent transcript from a triphosphate to a diphosphate by a RNA 5'-triphosphatase, followed by the addition of a guanosine monophosphate by the mRNA guanylyltransferase, to form a 5'-5'-triphosphate linkage. This cap is then methylated by 2'-O-methyltransferases.
Rossignol M, Kolb-Cheynel I, Egly JM.; ''Substrate specificity of the cdk-activating kinase (CAK) is altered upon association with TFIIH.''; PubMedEurope PMCScholia
Glover-Cutter K, Kim S, Espinosa J, Bentley DL.; ''RNA polymerase II pauses and associates with pre-mRNA processing factors at both ends of genes.''; PubMedEurope PMCScholia
Narita T, Yung TM, Yamamoto J, Tsuboi Y, Tanabe H, Tanaka K, Yamaguchi Y, Handa H.; ''NELF interacts with CBC and participates in 3' end processing of replication-dependent histone mRNAs.''; PubMedEurope PMCScholia
Görnemann J, Kotovic KM, Hujer K, Neugebauer KM.; ''Cotranscriptional spliceosome assembly occurs in a stepwise fashion and requires the cap binding complex.''; PubMedEurope PMCScholia
Bentley D.; ''Coupling RNA polymerase II transcription with pre-mRNA processing.''; PubMedEurope PMCScholia
Schultz P, Fribourg S, Poterszman A, Mallouh V, Moras D, Egly JM.; ''Molecular structure of human TFIIH.''; PubMedEurope PMCScholia
Heidemann M, Hintermair C, Voß K, Eick D.; ''Dynamic phosphorylation patterns of RNA polymerase II CTD during transcription.''; PubMedEurope PMCScholia
Yamada-Okabe T, Doi R, Shimmi O, Arisawa M, Yamada-Okabe H.; ''Isolation and characterization of a human cDNA for mRNA 5'-capping enzyme.''; PubMedEurope PMCScholia
Shatkin AJ, Manley JL.; ''The ends of the affair: capping and polyadenylation.''; PubMedEurope PMCScholia
Giglia-Mari G, Giglia-Mari G, Coin F, Ranish JA, Hoogstraten D, Theil A, Wijgers N, Jaspers NG, Raams A, Argentini M, van der Spek PJ, Botta E, Stefanini M, Egly JM, Aebersold R, Hoeijmakers JH, Vermeulen W.; ''A new, tenth subunit of TFIIH is responsible for the DNA repair syndrome trichothiodystrophy group A.''; PubMedEurope PMCScholia
Tsukamoto T, Shibagaki Y, Niikura Y, Mizumoto K.; ''Cloning and characterization of three human cDNAs encoding mRNA (guanine-7-)-methyltransferase, an mRNA cap methylase.''; PubMedEurope PMCScholia
Buratowski S.; ''Progression through the RNA polymerase II CTD cycle.''; PubMedEurope PMCScholia
Giacometti S, Benbahouche NEH, Domanski M, Robert MC, Meola N, Lubas M, Bukenborg J, Andersen JS, Schulze WM, Verheggen C, Kudla G, Jensen TH, Bertrand E.; ''Mutually Exclusive CBC-Containing Complexes Contribute to RNA Fate.''; PubMedEurope PMCScholia
Proudfoot NJ, Furger A, Dye MJ.; ''Integrating mRNA processing with transcription.''; PubMedEurope PMCScholia
At the beginning of this reaction, 1 molecule of 'RNA Pol II with phosphorylated CTD: CE complex' is present. At the end of this reaction, 1 molecule of 'RNA Pol II with phosphorylated CTD: CE complex with activated GT' is present.
At the beginning of this reaction, 1 molecule of 'mRNA capping enzyme', and 1 molecule of 'Pol II transcription complex with (ser5) phosphorylated CTD containing extruded transcript to +30' are present. At the end of this reaction, 1 molecule of 'RNA Pol II with phosphorylated CTD: CE complex' is present.
The capping enzyme interacts with the Spt5 subunit of transcription elongation factor DSIF. This interaction may couple the capping reaction with promoter escape or elongation, thereby acting as a "checkpoint" to assure that capping has occurred before the polymerase proceeds to make the rest of the transcript (Gonatopoulos-Pournatzis et al.2011).
The capping enzyme binds the 5'-end of the nascent transcript soon after it is synthesized on the DNA template, and results in the formation of the capping complex along with the C-terminal domain of RNA polymerase II, and Spt5 (Heidemann et al. 2013, Buratowski 2009, Schoenberg and Maquat 2009).
After the capping complex is formed, the RNA triphosphatase activity of the capping enzyme hydrolyzes the 5'-end phosphate group of the nascent mRNA transcript to a diphosphate. The RNA triphosphatase (RTP) domain of mammalian capping enzyme is a member of a superfamily of phosphatases that include the protein tyrosine phosphatases, some lipid phosphatases, and several nucleic acid phosphatases. This family uses a conserved nucleophilic cysteine residue to attack the target phosphate. A transient phospho-cysteinyl enzyme intermediate is then hydrolyzed to regenerate the enzyme active site. It should be noted that while higher eukaryotic capping enzymes use PTP-like triphosphatase domains, the yeast triphosphatases are a completely different class of enzymes. The yeast RTPs are metal-dependent phosphatases. RNA 5'-triphosphatase (RTP) catalyzed first reaction can be represented as:pppN(pN)n + GTP -> ppN(pN)n + Pi; (n=20-25)
A highly conserved lysine within the guanylyltransferase (GT) site of the mRNA capping enzyme attacks the alpha-phosphate of GTP. An enzyme-GMP covalent intermediate is formed.
The diphosphate 5'-end of the mRNA is joined to the GMP, releasing it from the enzyme. At this time, it is unclear how the RNA diphosphate end is transferred from the active site of the triphosphatase to the guanylyltransferase site. The covalent enzyme-GMP complex can form in the absence of RNA. Guanylyltransferase (GT) catalyzed second reaction can be represented as:ppN(pN)n + GTP -> GpppN(pN)n + PPi
In the final step of the capping reaction, the methyltransferase takes a methyl group from S-adenosyl-methionine to the N7 position of the cap guanine. N7G-methyltransferase (MT) mediated reaction can be represented as: GpppN(pN)n + S-adenosylmethionine (Adomet) ->m7GpppN(pN)n + S-adenosylhomocysteine (Adohcy).
At the beginning of this reaction, 1 molecule of 'CBP80', and 1 molecule of 'CBP20' are present. At the end of this reaction, 1 molecule of 'Cap Binding Complex (CBC)' is present (Glover-Cutter et al., 2008, Görnemann et al., 2005, Narita et al., 2007).
This reaction takes place in the nucleus.
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DataNodes
(with freed 5'-
GMP)complex with (ser5) phosphorylated CTD containing extruded
transcript to +30Polymerase II
(phosphorylated):TFIIF:capped pre-mRNAphosphorylated CTD: CE complex with
activated GTphosphorylated CTD:
CE complexpre-mRNA:CBC:RNA Pol II (phosphorylated)
complexAnnotated Interactions
(with freed 5'-
GMP)(with freed 5'-
GMP)(with freed 5'-
GMP)complex with (ser5) phosphorylated CTD containing extruded
transcript to +30This reaction takes place in the 'nucleus'.
This reaction takes place in the 'nucleus'.
The RNA triphosphatase (RTP) domain of mammalian capping enzyme is a member of a superfamily of phosphatases that include the protein tyrosine phosphatases, some lipid phosphatases, and several nucleic acid phosphatases. This family uses a conserved nucleophilic cysteine residue to attack the target phosphate. A transient phospho-cysteinyl enzyme intermediate is then hydrolyzed to regenerate the enzyme active site. It should be noted that while higher eukaryotic capping enzymes use PTP-like triphosphatase domains, the yeast triphosphatases are a completely different class of enzymes. The yeast RTPs are metal-dependent phosphatases. RNA 5'-triphosphatase (RTP) catalyzed first reaction can be represented as:pppN(pN)n + GTP -> ppN(pN)n + Pi; (n=20-25)
Guanylyltransferase (GT) catalyzed second reaction can be represented as:ppN(pN)n + GTP -> GpppN(pN)n + PPi
(Yamada-Okabe et al. 1998).
GpppN(pN)n + S-adenosylmethionine (Adomet) ->m7GpppN(pN)n + S-adenosylhomocysteine (Adohcy).
Polymerase II
(phosphorylated):TFIIF:capped pre-mRNAPolymerase II
(phosphorylated):TFIIF:capped pre-mRNAphosphorylated CTD: CE complex with
activated GTphosphorylated CTD: CE complex with
activated GTphosphorylated CTD:
CE complexphosphorylated CTD:
CE complexpre-mRNA:CBC:RNA Pol II (phosphorylated)
complex