The Cytoplasmic Trancriptase Complex

A rather unique RNA polymerase, a DExH/D box helicase and an mRNA capping enzyme, represent key elements of the cytoplasmic transcriptional apparatus. Consistent with their pivotal function, each of the above genes were proven essential for plasmid maintenance (Schaffrath et al. 1995, 1997; Larsen et al. 1998).

The RNA polymerase (Fig. 3) appears to consist of two different subunits, encoded by pGKL2 ORF6 and ORF7, and homologous genes in other plasmid systems. The architecture of the enzyme resembles ß and ß'-subunits of the multisubunit Escherichia coli RNA polymerase; all ß and several domains of the ß' subunit exist in the predicted pGKL2/Orf6 protein (Wilson and Mea-cock 1988; Thuriaux and Sentenac 1992; Schaffrath et al. 1995). However, two ß' domains, located usually close to the C terminus, reside in a separately encoded protein (pGKL2 Orf7p or its homologues; Schaffrath et al. 1997; Jeske et al. 2006a), suggesting ß' functions can be assigned to separate subunits, a scenario known for the archeon Halobacterium halobium (Leffers et al. 1989). Thus, concerning its domain architecture the linear plas-mid encoded RNA polymerase displays a rather unique and simple structure, most closely related to multisubunit RNA polymerases, in marked contrast to the single subunit, T7-like RNA polymerase encoded by mitochondrial linear plasmids.

The predicted protein encoded by pGKL2 ORF4 (and respective homologues in other autonomous linear plasmids) belongs to the DExH/D box family which comprises RNA helicases and NTPases (Tommasino et al. 1988; Stark et al. 1990; Jeske et al. 2006a). Though ORF4 was proven to be essential for plasmid maintenance (Schaffrath et al. 1997), the precise function of the predicted polypeptide remains to be elucidated. There are (at least) 34 DExD/H helicase homologues in Saccharomyes cerevisiae, most of which are essential (de la Cruz et al. 1999). Members of this family are instrumental in a number of diverse processes related to RNA metabolism (such as ribosome biogenesis, mRNA splicing, RNA degradation, nuclear export) as well as transcription and translation (de la Cruz et al. 1999; Tanner and Linder 2001). Not all representatives assigned to such a family must necessarily possess helicase activity but may rather supply energy for other processes (by NTP hydrolysis), such as the disruption of ribonucleoprotein complexes (Schwer 2001; Jankowsky and Bowers 2006). Interestingly, vaccinia virus NphI, a DExH/D protein, displays ATPase but lacks helicase activity. The enzyme, stimulated by ssDNA, acts as an energy-coupling factor for transcription elongation and mRNA release upon termination (Deng and Shuman 1998). Be that as it may, though the role of the DExH/D protein encoded by pGKL2 ORF4 systems remains obscure at present, it is likely to be involved in the rather unique cytoplasmic transcription process.

An unambiguously essential component of the cytoplasmic transcription machinery is the mRNA capping enzyme (encoded by pGKL2, ORF3, and homologous genes in other systems) (Larsen et al. 1998; Tiggemann et al. 2001; Klassen et al. 2001; Jeske and Meinhardt 2006). Orf3p displays RNA triphosphatase and guanylyltransferase activities (Tiggemann et al. 2001). During mRNA cap formation, RNA triphosphatase removes the y-phosphate from the 5' end of the nascent mRNA, followed by formation of an unusual 5'-5' linkage between guanosine monophosphate and the processed 5' transcript end by the guanylyltransferase, in a reaction that involves a covalent intermediate between a lysine residue of the enzyme and GMP. Finally, the cap methyltransferase modifies the guanosine residue at position N7 utilizing S-adenosylmethionine (SAM) as the methyl donor (Martin et al. 1975; Shu-man and Hurwitz 1981; Cong and Shuman 1993; Shuman and Schwer 1995; Bisaillon and Lemay 1997). In contrast to RNA triphosphatase and guanylyl-transferase activities, cap methyltransferase activity was not obtained for the purified Orf3p, though there is a potential SAM binding site (Larsen et al. 1998; Tiggemann et al. 2001). Overall, Orf3p resembles the capping enzyme known from the cytoplasmic vaccinia virus, in which triphosphatase, guany-lyltransferase, and methyltransferase domains are located in the polypeptide (vD1) encoded by the vaccinia D1 gene. However, vD1 displays only faint cap methyltransferase activity, which is stimulated tremendously (more than 30fold) upon heterodimerization with the vaccinia D12 gene product (Cong and Shuman 1992; Higman et al. 1992, 1994; Mao and Shuman 1994). It remains to be elucidated, however, whether the methyltransferase activity of the linear plasmid encoded capping enzyme (Orf3p) requires a stimulatory subunit, too. Concerning domain architecture and sequence similarities, the linear plasmid encoded Orf3p clearly corresponds to capping enzymes of irido-and poxviridae, which differ strikingly from fungal nuclearly encoded capping enzymes as—in the latter—RNA triphosphatase, guanylyltransferase, and cap methyltransferase activities reside in separate polypeptides. The persuasive similarity of the mRNA capping machinery of cytoplasmic linear plasmids to that of cytoplasmic pox viruses again strongly supports the conclusive presumption for viral ancestry.

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