Cytoplasmic Transcription

Since the cytoplasmic transcriptase complex in general, and the RNA poly-merase in particular, convincingly differs from the nuclearly encoded host enzyme, cytoplasmic transcription of linear plasmid based genes was—not astonishingly—shown to be driven by unique promoters clearly differing from nuclear ones (Kämper et al. 1989a,b, 1991; Gunge et al. 2003; Romanos and Boyd 1988; Stark et al. 1990). Based on cytoplasmic in vivo recombination approaches using either yeast nuclear or reporter genes of bacterial origin fused to pGKLl and pGKL2 sequences, a 6-bp spanning conserved motif preceding the native ORFs was defined as being necessary and sufficient for cytoplasmic transcription (upstream conserved sequence, UCS: 5'-ATNTGA-3'; Schründer and Meinhardt 1995; Schaffrath et al. 1996; Schickel et al. 1996; Schründer et al. 1996). The UCS motif is typically located approximately 30 bp upstream of the start codon of an ORF; however, distances may vary. Sequences distal of the UCSs were found to be irrelevant, ruling out additional promoter motifs (Schickel et al. 1996).

Consistent with the high degree of conservation of transcriptase complexes, UCS motifs are located upstream of every ORF in any cytoplasmic plasmid system, irrespective of the host (Bolen et al. 1994; Hishinuma and Hirai 1991; Fukuda et al. 1997; Klassen et al. 2001, 2002, 2004; Klassen and Meinhardt 2003; Jeske and Meinhardt 2006). Accordingly, a UCS derived from the D. etchellsii plasmid pPE1B was shown to act as a functional promoter in the K. lactis pGKL system (Klassen et al. 2001), concomitantly providing further evidence for a functionally conserved cytoplasmic transcriptase.

Transcription initiation, analyzed for a number of ORFs in the pGKL system, occurs at multiple sites, routinely around 8-16 bp downstream of the UCS motif, with a preference for one site (Romanos and Boyd 1988; Jeske et al. 2006b). Since Northern analyses revealed monocistronic transcripts only, defined termination of transcripts was suggested to occur (Schaffrath et al. 1995; Jeske et al. 2006b). To date, however, the termination mechanism remains totally obscure, as for the potential 3' modification such as polyadenylation.

Though there are similarities between a potential DExH/D box ATPase (Orf4p) encoded by linear plasmids and the viral NphI proteins being instrumental in transcription termination, it has not been proven that this largely uncharacterized enzyme functions similarly in linear plasmid systems.

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