ELOF1 is a core component of the promoter-proximal paused RNA polymerase II complex

Metazoan RNA polymerase II is prone to pause in the promoter-proximal region of genes. This pausing involves DRB Sensitivity-Inducing Factor (DSIF) and Negative Elongation Factor (NELF). DSIF and NELF, however, are insufficient to fully reconstitute the degree of promoter-proximal pausing observed in cells. Their negative effects are counteracted by transcription initiation factor TFIIF or by physiological nucleotide concentrations, indicating an additional factor is required. Here, we report that Elongation Factor Homolog 1 (ELOF1) is this missing factor. ELOF1 is enriched in the promoter-proximal region of genes, and its rapid degradation reduces pause duration in cells. In reconstituted assays, ELOF1 potently enhances pausing induced by DSIF and NELF at physiological nucleotide concentrations. Cryo-EM structures reveal that DSIF-NELF-ELOF1 sterically clashes with the position of TFIIF on RNA polymerase II. Accordingly, RNA polymerase II-DSIF-NELF-ELOF1, but not RNA polymerase II-DSIF-NELF, counteracts the positive effects of TFIIF. Our results establish ELOF1 as a core component of promoter-proximal paused RNA polymerase II.