%0 Journal Article %A Irina O. Vvedenskaya %A Jeremy G. Bird %A Yuanchao Zhang %A Yu Zhang %A Xinfu Jiao %A Ivan Barvík %A Libor Krásný %A Megerditch Kiledjian %A Deanne M. Taylor %A Richard H. Ebright %A Bryce E. Nickels %T “CapZyme-Seq” comprehensively defines promoter-sequence determinants for RNA 5’ capping with NAD+ %D 2017 %R 10.1101/239426 %J bioRxiv %P 239426 %X 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. %U https://www.biorxiv.org/content/biorxiv/early/2017/12/24/239426.full.pdf