Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters

RNA polymerases are highly regulated molecular machines. We present a method (GRO-seq) that maps the position, amount, and orientation of transcriptionally-engaged RNA polymerases genome-wide. In this method, nuclear run-on RNAs are subjected to large-scale parallel sequencing and mapped to the genome. Here, we show that peaks of promoter-proximal polymerase reside on ~30% of human genes, transcription extends beyond pre-mRNA 3’ cleavage, and antisense transcription is prevalent. Additionally, most promoters have an engaged polymerase upstream and in an orientation opposite to the annotated gene. This divergent polymerase is associated with active genes, but does not elongate effectively beyond the promoter. These results imply that the interplay between polymerases and regulators over broad promoter regions dictates the orientation and efficiency of productive transcription. Overall design: Two biological replicates of nascent RNA sequencing