| mRNA guanylyltransferase | |||||||||
|---|---|---|---|---|---|---|---|---|---|
 | |||||||||
| Identifiers | |||||||||
| EC no. | 2.7.7.50 | ||||||||
| CAS no. | 56941-23-2 | ||||||||
| Databases | |||||||||
| IntEnz | IntEnz view | ||||||||
| BRENDA | BRENDA entry | ||||||||
| ExPASy | NiceZyme view | ||||||||
| KEGG | KEGG entry | ||||||||
| MetaCyc | metabolic pathway | ||||||||
| PRIAM | profile | ||||||||
| PDB structures | RCSB PDBPDBePDBsum | ||||||||
| |||||||||
Acapping enzyme (CE) is anenzyme thatcatalyzes the attachment of the5' cap tomessenger RNA molecules that are in the process of being synthesized in thecell nucleus during the first stages ofgene expression. The addition of the cap occursco-transcriptionally, after the growingRNA molecule contains as little as 25nucleotides. The enzymatic reaction is catalyzed specifically by thephosphorylated carboxyl-terminal domain (CTD) ofRNA polymerase II. The 5' cap is therefore specific to RNAs synthesized by this polymerase rather than those synthesized byRNA polymerase I orRNA polymerase III.Pre-mRNA undergoes a series of modifications - 5' capping, splicing and 3'polyadenylation before becoming mature mRNA that exits thenucleus to be translated into functional proteins and capping of the 5' end is the first of these modifications. Three enzymes,RNA triphosphatase,guanylyltransferase (or CE), andmethyltransferase are involved in the addition of the methylated 5' cap to the mRNA.
Capping is a three-step process that utilizes theenzymes RNA triphosphatase, guanylyltransferase, and methyltransferase.[1][2] Through a series of three steps, the cap is added to the first nucleotide's 5' hydroxyl group of the growingmRNA strand whiletranscription is still occurring.[1][3] First, RNA 5' triphosphatase hydrolyzes the 5' triphosphate group to make diphosphate-RNA. Then, the addition ofGMP by guanylyltransferase produces theguanosine cap. Last, RNA methyltransferase transfers amethyl group to the guanosine cap to yield 7-methylguanosine cap that is attached to the 5' end of the transcript.[1][3][4][5] These three enzymes, collectively called the capping enzymes, are only able tocatalyze their respective reactions when attached to RNA polymerase II, an enzyme necessary for the transcription of DNA into pre-mRNA. When this complex of RNA polymerase II and the capping enzymes is achieved, the capping enzymes are able to add the cap to the mRNA while it is produced by RNA polymerase II.[6]
Eukaryotic RNA must undergo a series of modifications in order to be exported from thenucleus and successfully translated into function proteins, many of which are dependent on mRNA capping, the first mRNA modification to take place.[6][7] 5' capping is essential for mRNA stability, enhancing mRNA processing, mRNA export and translation.[1][7][8] 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.[6] The addition of the cap onto mRNA confers protection to the transcript from exonucleases that degrade unprotected RNA and assist in the nuclear export transport process so that the mRNA can be translated to form proteins.[1] The function of the 5' cap is essential to the ultimate expression of the RNA.[1]
The capping enzyme is part of the covalentnucleotidyl transferasessuperfamily, which also includesDNA ligases andRNA ligases.[7][9][10][11] The enzymes of this superfamily share the following similarities:
The capping enzyme is composed of twodomains, a nucleotidyl transferase (NTase) domain and a C-terminal oligonucleotide binding (OB) domain.[7][10] The NTase domain, conserved in capping enzymes, DNA and RNA ligases, is made up 5 motifs, I, III, IIIa, IV and V.[7][10] Motif I or KxDG is the active site where the covalent (lysyl)-N-GMP intermediate is formed.[7][8][9][11] Both the NTase and OB domains undergo conformational changes that assist in the capping reaction.[10]
Capping enzymes are found in thenucleus ofeukaryoticcells.[8][12] Depending on the organism, the capping enzyme is either a monofunctional or bifunctionalpolypeptide.[4][5] The guanylyltransferases (Ceg1) ofSaccharomyces cerevisiae is encoded by theCEG1 gene and is composed of 459 amino acids (53-kD).[4][13] The RNA triphosphatase (Cet1) is a separate 549 amino acidpolypeptide (80-kD), encoded by theCET1 gene.[4][13][14] The human capping enzyme is an example of a bifunctional polypeptide, which has both triphosphatase (N-terminal) and guanylyltransferase (C-terminal) domains.[15][16] The humanmRNA guanylyltransferase domain of the capping enzyme is composed of sevenhelices and fifteenβ strands that are grouped into three, five and seven strands, arranged as antiparallelβ sheets.[15] The enzyme structure has three sub-domains referred to hinge, base and lid.[15] TheGTP binding site is located between the hinge and base domain.[15] The lid domain determines the conformation of theactive site cleft, which consists of the GTP binding site, phosphoamide linking lysine and surrounding residues.[15] The guanylyltransferase domain is linked to the triphosphatase domain via a 25 amino acid flexible loop structure.[15]
Splicing is dependent on the presence of the 7-methylguanosine cap. A defect in splicing can occur as a result of mutation(s) in theguanylyltransferase, which can inhibit enzyme activity, preventing the formation of the cap. However the severity of the effect is dependent on the guanylyltransferase mutation.[1] Furthermore, the guanylyltransferase relieves transcriptional repression mediated byNELF.[1][17] NELF together withDSIF prevents transcription elongation.[1][5] Thus, mutations in the enzyme can affect transcription elongation.[1]