Mechanism of the mRNA guanylyltransferase reaction: isolation of N epsilon-phospholysine and GMP (5′ leads to N epsilon) lysine from the guanylyl-enzyme intermediate.

Abstract

The mRNA capping reaction catalyzed by rat liver mRNA guanylyltransferase proceeds through an enzyme‐GMP intermediate in which GMP is linked to the enzyme by a phosphoamide linkage. The studies described here show that GMP is bound to the epsilon‐amino group of lysine of rat liver guanylyltransferase. The enzyme‐[32P]GMP intermediate was digested with pronase to a [32P]GMP‐peptide which was then converted to [32P]phosphoryl‐peptide through periodate oxidation followed by beta‐elimination. After alkaline hydrolysis of the [32P]phosphoryl‐peptide, the major radioactive product co‐electrophoresed with the authentic N epsilon‐phospholysine on DEAE‐cellulose paper. Neither [32P]Nimid‐phosphohistidine nor Nguanido‐phosphoarginine was detected in the hydrolysates. Furthermore, formation of N epsilon‐guanylyl‐lysine linkage on the enzyme was more directly shown by isolation of [32P]GMP(5′ leads to N epsilon)lysine when the steps of periodate oxidation and beta‐elimination were omitted. The results indicate that the nucleophile in the guanylyltransferase to which the guanylyl residue is linked is the epsilon‐amino group of a lysine residue. [32P]Phosphoryl‐lysine was also isolated from the vaccinia virus capping enzyme‐[32P]GMP intermediate. Guanylyltransferase from HeLa cells, wheat germ, Artemia salina and yeast also formed the enzyme‐GMP complex and, from the stability of the complex, the linkage between the enzyme and GMP was suggested to be a phosphoamide.

Demonstration of enzyme activities required for cap structure formation in infectious bursal disease virus, a member of the birnavirus group

“…In vaccinia virus, it has been shown that this transguanylylation catalysed by the vaccinia virus capping enzyme involves the formation of a covalent enzyme-guanylate intermediate (Shuman & Hurwitz, 1981). Formation of such intermediates has been identified in guanylyltransferases isolated from a number of organisms, including, among others, HeLa cells (Venkatesan & Moss, 1982;Shuman, 1982;Wang et al, 1982), rat liver (Muzimoto et al, 1982;Toyama et al, 1983), Artemia salina, wheatgerm and yeast (Toyama et al, 1983). Apparently a similar mechanism also mediates the guanylyltransferase reaction of reovirus (Shatkin et al, 1983;Zarbl & Millward, 1983) and, as shown here, the birnavirus IBDV.…”

The coat protein of the yeast double-stranded RNA virus L-A attaches covalently to the cap structure of eukaryotic mRNA.

“…Lane 1 of Fig. 10A 50 ,uM PPi during the reaction had no effect on the amount of linkage, ruling out a mechanism that was described for cellular and viral capping enzymes, whereby a covalent enzyme-GMP intermediate is generated concomitantly with a release of PP; (48,54). To examine the number of sites on the protein at which linkage occurs, RNA-linked L-A coat protein was trypsin digested, and the peptides were subjected to two-dimensional analysis (Fig.…”

Specific in vitro guanylylation of a 43-kilodalton membrane-associated protein of Streptomyces coelicolor

“…These properties (i.e., acid lability and alkaline stability) as well as the resistance of the complex to hydrolysis by snake venom phosphodiesterases suggest a phosphoamide linkage between GMP and the protein (4). The cleavage of the complex by acidic hydroxylamine (pH 4.75) also strongly supports a phosphoamide linkage, such as the GMP(5'-Ne) lysine found in rat liver guanylyltransferase (21) and T4 RNA ligase (6). For bacteria, there are two reports of apparent guanylylation in proteins in B. subtilis (11,12).…”

“…Such is the case in the RNA capping enzyme from vaccinia virus, in which an enzymeguanylylated intermediate necessary for the synthesis of cap structures is formed (16). Covalent enzyme-GMP intermediate complexes have also been identified in guanylyltransferases isolated from HeLa cells (17), rat liver, wheat germ, Artemia salina, and Saccharomyces cerevisiae (21). Recently, guanylylation of the major regulatory protein of herpes simplex virus has been described (1).…”