The study introduces a novel method called Global Run-On Sequencing (GRO-seq) to map and quantify the position, amount, and orientation of transcriptionally engaged RNA polymerases across the human genome. GRO-seq uses nuclear run-on RNA molecules, which are subjected to large-scale parallel sequencing and mapped to the genome. Key findings include: 1. **Promoter-Proximal Pausing**: Peaks of polymerase occupancy are observed near ~30% of human genes, indicating promoter-proximal pausing. 2. **Transcription Beyond 3' CLEavage**: Transcription extends beyond the pre-mRNA 3' cleavage site. 3. **Antisense Transcription**: Antisense transcription is prevalent in ~58.7% of genes. 4. **Divergent Polymerase Orientation**: Most promoters have an engaged polymerase upstream and in the opposite orientation to the annotated gene, which is associated with active genes but does not effectively elongate beyond the promoter. 5. **Correlation with Gene Activity**: The density of polymerases within the promoter-proximal region correlates with gene activity, and the pausing index inversely correlates with gene activity. 6. **Divergent Transcription**: A significant number of genes (55%) display divergent transcription within 1 kb upstream of sense-oriented promoter-proximal peaks, indicating that the number of bidirectional promoters exceeds previous estimates. 7. **CpG Island Correlation**: Gene activity, pausing, and divergent transcription correlate with promoters containing CpG islands. These findings suggest that the interplay between polymerases and regulators over broad promoter regions dictates the orientation and efficiency of productive transcription. The study also discusses potential functions of divergent transcription, such as facilitating access to control elements and creating barriers to prevent nucleosome obstruction.The study introduces a novel method called Global Run-On Sequencing (GRO-seq) to map and quantify the position, amount, and orientation of transcriptionally engaged RNA polymerases across the human genome. GRO-seq uses nuclear run-on RNA molecules, which are subjected to large-scale parallel sequencing and mapped to the genome. Key findings include: 1. **Promoter-Proximal Pausing**: Peaks of polymerase occupancy are observed near ~30% of human genes, indicating promoter-proximal pausing. 2. **Transcription Beyond 3' CLEavage**: Transcription extends beyond the pre-mRNA 3' cleavage site. 3. **Antisense Transcription**: Antisense transcription is prevalent in ~58.7% of genes. 4. **Divergent Polymerase Orientation**: Most promoters have an engaged polymerase upstream and in the opposite orientation to the annotated gene, which is associated with active genes but does not effectively elongate beyond the promoter. 5. **Correlation with Gene Activity**: The density of polymerases within the promoter-proximal region correlates with gene activity, and the pausing index inversely correlates with gene activity. 6. **Divergent Transcription**: A significant number of genes (55%) display divergent transcription within 1 kb upstream of sense-oriented promoter-proximal peaks, indicating that the number of bidirectional promoters exceeds previous estimates. 7. **CpG Island Correlation**: Gene activity, pausing, and divergent transcription correlate with promoters containing CpG islands. These findings suggest that the interplay between polymerases and regulators over broad promoter regions dictates the orientation and efficiency of productive transcription. The study also discusses potential functions of divergent transcription, such as facilitating access to control elements and creating barriers to prevent nucleosome obstruction.