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

SUMMARYNucleoside-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 byEscherichia coliRNAP 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 toE. colitotal cellular RNA, we establish that sequence determinants for NCIN cappingin vivomatch the NAD+-capping consensus definedin 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 cappingin vitroandin vivo.