Question

Describe the mechanism for how the 5’ to 5’ triphosphate linkage is formed between the GTP and the first mRNA nucleotide.

Answer 1

The 5’ to 5’ triphosphate linkage between the 5’ end of mRNA (first mRNA nucleotide) and GTP occurs during 5’ Capping. In 5’ capping of eukaryotic mRNA, the 7-methylguanosine cap is joined to first nucleotide, via a triphosphate linkage.

Initially a 5’ triphosphate on mRNA is hydrolyzed to remove the gamma phosphate to form diphosphate RNA (containing alpha and beta phosphate). There is release of gamma phosphate. This reaction is carried out by RNA 5’ triphosphatase enzyme.

The alpha phosphate of GTP will first react with another enzyme guanlyltransferase enzyme via the lysine residue of the enzyme. This leads to formation of guanylyl transferase-GMP intermediate (linkage via the lysine of teh enzyme), and pyrophosphate is released. This enzyme-GMP intermediate catalyzes the next step of the reaction. The enzyme transfers the GMP to diphosphate RNA. This results in formation of a 5’ -5’ linkage between beta phosphate of mRNA first nucleotide and alpha phosphate of GMP. The guanylyl transferase enzyme is thus regenerated.

The final step of capping is catalyzed by the RNA (guanine-7- ) methyltransferase enzyme. The RNMT enzyme will add methyl group from S-adenosylmethionine (SAM) to the guanosine cap. This methyl group is added at the N-7 position of guanosine, resulting in formation of the 7- methylguanosine cap. Another enzyme 7 methylguanosine-specific 2′O methyltransferase (2'O MTase) will add another methyl group to the first nucleotide of mRNA to form the final cap. This methyl group is transferred from SAM to the 2’ oxygen position of ribose sugar of the RNA nucleotide. Thus, first methyl group is added to Guanosine and the other methyl group is added to nucleotide.