Abstrakt
Nucleoside-containing metabolites such asNAD(+) can be incorporated as 50 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 similar to 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.