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Krasny, L.

Publications and source records attributed to Krasny, L..

2 recordsLinked to original sources

Transcription apparatus of the yeast killer DNA plasmids: Architecture, function, and evolutionary origin

Transcription of extrachromosomal elements such as organelles, viruses, and plasmids is dependent on cellular RNA polymerase (RNAP) or intrinsic RNAP encoded by these elements. The yeast Kluyveromyces lactis contains killer DNA plasmids that bear putative non-canonical RNAP genes. Here, we describe the architecture and evolutionary origin of this transcription machinery. We show that the two RNAP subunits interact in vivo, and this complex interacts with another two plasmid-encoded proteins - the mRNA capping enzyme, and a putative helicase which interacts with plasmid-specific DNA. Further, we identify a promoter element that causes 5 polyadenylation of plasmid-specific transcripts via RNAP slippage during transcription initiation, and structural elements that precede the termination sites. As a result, we present a first model of the yeast killer plasmid transcription initiation and intrinsic termination. Finally, we demonstrate that plasmid RNAP and its promoters display high similarity to poxviral RNAP and promoters of early poxviral genes, respectively.

molecular biology

“CapZyme-Seq” comprehensively defines promoter-sequence determinants for RNA 5’ capping with NAD+

Nucleoside-containing metabolites such as NAD+ can be incorporated as \"5' caps\" on RNA by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase (RNAP). Here, we report \"CapZyme-Seq,\" a high-throughput-sequencing method that employs NCIN-decapping enzymes NudC and Rai1 to detect and quantify NCIN-capped RNA. By combining CapZyme-Seq with multiplexed transcriptomics, we determine efficiencies of NAD+ capping by Escherichia coli RNAP for ~16,000 promoter sequences. The results define preferred transcription start-site (TSS) positions for NAD+ capping and define a consensus promoter sequence for NAD+ capping: HRRASWW (TSS underlined). By applying CapZyme-Seq to E. coli total cellular RNA, we establish that sequence determinants for NCIN capping in vivo match the NAD+-capping consensus defined in vitro, and we identify and quantify NCIN-capped small RNAs. Our findings define the promoter-sequence determinants for NCIN capping with NAD+ and provide a general method for analysis of NCIN capping in vitro and in vivo.

biochemistry