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Structural biology of mammalian transcription regulation

Periodic Reporting for period 3 - TRANSREGULON (Structural biology of mammalian transcription regulation)

Reporting period: 2019-07-01 to 2020-06-30

The ERC-funded research project TRANSREGULON addresses the question of how mammalian genes are regulated, i.e. how they are switched on or off in cells when needed. Genes are copied (transcribed) by RNA polymerase, an enzyme that uses DNA as a template to produce RNA, which in turn serves as a building plan for proteins. RNA polymerase often pauses near the beginning of genes and such pausing can down-regulate gene transcription. The enzyme is then released and activated by positive cellular signals. During this project we provided major new insights into the molecular mechanism of RNA polymerase pausing and its activation. This was achieved by combining several techniques of molecular and structural biology. Since the dysregulation of genes occurs during many diseases, including cancer, it was of great biomedical importance and interest to the society to understand the mechanisms of gene regulation uncovered during this work.
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.
By the end of the project, we have now achieved a comprehensive molecular understanding of how transcription is regulated at the beginning of genes near the gene promoter. We have visualized how negative factors make the RNA polymerase pause and how positive factors help to release this negative impact and activate the polymerase for transcription elongation. The obtained understanding represents great progress beyond the state of the art and took the research field of gene regulation to a new level.