Abstract
Extrachromosomal hereditary elements such as organelles, viruses, and plasmids are important for the cell fitness and survival. Their transcription is dependent on host cellular RNA polymerase (RNAP) or intrinsic RNAP encoded by these elements. The yeast Kluyveromyces lactis contains linear cytoplasmic DNA virus-like elements (VLEs, also known as linear plasmids) that bear genes encoding putative non-canonical two-subunit RNAP. Here, we describe the architecture and identify the evolutionary origin of this transcription machinery. We show that the two RNAP subunits interact in vivo, and this complex interacts with another two VLE-encoded proteins, namely the mRNA capping enzyme and a putative helicase. RNAP, mRNA capping enzyme and the helicase also interact with VLE-specific DNA in vivo. Further, we identify a promoter sequence element that causes 5′ mRNA polyadenylation of VLE-specific transcripts via RNAP slippage at the transcription initiation site, and structural elements that precede the termination sites. As a result, we present a first model of the yeast virus-like element transcription initiation and intrinsic termination. Finally, we demonstrate that VLE RNAP and its promoters display high similarity to poxviral RNAP and promoters of early poxviral genes, respectively, thereby pointing to their evolutionary origin.
Highlights
Linear double-stranded DNA virus-like elements (VLEs) were found in the cytoplasm of several yeast species
We re-opened investigation of these elements focused on considerably underexplored nucleus-independent transcription of K. lactis VLEs
We identified new DNA and RNA elements that were directly connected with formation of VLE-specific mRNA ends, as demonstrated by mutagenesis of these elements and its effect on VLE transcription in vivo
Summary
Linear double-stranded DNA virus-like elements (VLEs) were found in the cytoplasm of several yeast species. Yeast linear plasmids of Kluyveromyces lactis, termed pGKL1 (or K1) and pGKL2 (or K2), have become a model system to study such DNA elements These VLEs have compact genomes with occasional overlaps of open reading frames (ORFs) and a high AT content of ~74% [5, 6]. The presence of both pGKL plasmids in several K. lactis strains is associated with the extensively studied yeast killer phenotype [7]
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