Transcription apparatus of the yeast virus-like elements: Architecture, function, and evolutionary origin

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.

细胞器、病毒、质粒等染色体外遗传元件，对于细胞适应性与存活至关重要。这类元件的转录依赖于宿主细胞RNA聚合酶（RNAP），或是由其自身编码的内源性RNAP。乳酸克鲁维酵母（Kluyveromyces lactis）中存在线型细胞质DNA病毒样元件（VLEs，亦称线型质粒），该元件携带有编码假定非经典双亚基RNAP的基因。本研究阐明了该转录机器的结构组成，并解析了其进化起源。我们证实，这两个RNAP亚基可在体内发生相互作用；该复合物还可与另外两种由VLE编码的蛋白——即mRNA加帽酶与假定解旋酶——发生互作。RNAP、mRNA加帽酶与该解旋酶同样可在体内与VLE特异性DNA结合。此外，我们鉴定到一种启动子序列元件，该元件可通过转录起始位点处的RNAP滑动，导致VLE特异性转录本的5'端mRNA多聚腺苷酸化；同时还鉴定到位于终止位点上游的结构元件。据此，我们首次提出了该酵母病毒样元件的转录起始与内源性终止模型。最后，我们证实VLE RNAP及其启动子分别与痘病毒RNAP及痘病毒早期基因启动子具有高度同源性，由此揭示了其进化来源。