We proposed to use an integrated structural biology approach combining X-ray crystallography, cryo-electron microscopy, and crosslinking, to obtain the first atomic structure of a mammalian Pol II enzyme (aim 1), the structure of the negative elongation factor NELF (aim 2), which is required for promoter-proximal pausing by mammalian Pol II, and the three-dimensional architecture of the promoter-proximally paused Pol II complex (aim 3) containing the multiprotein pausing factors NELF and DSIF. We have achieved all three aims in full. With respect to aim 1, we were able to establish the purification protocol for mammalian RNA polymerase II (Pol II) and could solve the 3.4-Å-resolution cryo-electron microscopy structure of mammalian Pol II in the form of a transcribing complex comprising DNA template and RNA transcript. With respect to aim 2, we were able to crystallize and report the crystal structure of the subcomplex of NELF consisting of part of subunit NELF-A and part of subunit NELF-C. With respect to aim 3, we could solve the structure of the mammalian Pol II elongation complex with bound factor DSIF, and the structure of the paused transcription complex Pol II-DSIF-NELF. We have additionally solved the structure of the released and activated transcription complex Pol II-DSIF-PAF-SPT6. Together this work provided incredible new insights into how the gene switch near promoters works.