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 (sRNAs). 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.

Department of Cell Biology and Neuroscience Rutgers University Piscataway NJ 08854 USA

Department of Chemistry and Waksman Institute Rutgers University Piscataway NJ 08854 USA

Department of Genetics and Waksman Institute Rutgers University Piscataway NJ 08854 USA

Department of Genetics and Waksman Institute Rutgers University Piscataway NJ 08854 USA; Department of Biomedical and Health Informatics Children's Hospital Philadelphia PA 19041 USA

Department of Genetics and Waksman Institute Rutgers University Piscataway NJ 08854 USA; Department of Biomedical and Health Informatics Children's Hospital Philadelphia PA 19041 USA; Department of Pediatrics Perelman School of Medicine University of Pennsylvania Philadelphia PA 19104 USA; Department of Obstetrics Gynecology and Reproductive Sciences Rutgers Robert Wood Johnson Medical School New Brunswick NJ 08901 USA

Department of Genetics and Waksman Institute Rutgers University Piscataway NJ 08854 USA; Department of Chemistry and Waksman Institute Rutgers University Piscataway NJ 08854 USA

Division of Biomolecular Physics Institute of Physics Faculty of Mathematics and Physics Charles University Ke Karlovu 5 12116 Prague 2 Czech Republic

Institute of Microbiology Czech Academy of Sciences v v i Vídeňská 1083 14220 Prague 4 Czech Republic

Key Laboratory of Synthetic Biology CAS Center for Excellence in Molecular Plant Sciences Institute of Plant Physiology and Ecology Chinese Academy of Sciences Shanghai 200032 China

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