Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - TRANSREGULON (Structural biology of mammalian transcription regulation)

Teaser

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...

Summary

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. During this project we provide major new insights into the molecular mechanism of RNA polymerase pausing at the beginning of genes. This is achieved by combining several techniques of molecular and structural biology. Since the dysregulation of genes occurs during many diseases, including cancer, it is of great biomedical importance and interest to the society to understand the mechanisms of gene regulation uncovered during this work.

Work performed

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. Until now, we have achieved aims 1 and 2 and also a large part of aim 3. 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-Ã…-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. We now also solved the structures of the paused transcription complex Pol II-DSIF-NELF and the structure of the release and activated transcription complex Pol II-DSIF-PAF-SPT6. Together this work provides incredible new insights into how the gene switch near promoters works.

Final results

By the end of the project, we expect to have achieved a comprehensive molecular understanding of how transcription is regulated at the beginning of genes. We hope to have visualized how negative factors make the RNA polymerase pause and how positive factors help to release this negative impact and activate the polymerase. We will now concentrate on the complete, atomic structure of the released and activated Pol II enzyme, to uncover the roles that the various elongation factors play in activating Pol II. Such an understanding will represent great progress beyond the state of the art and will take the research field of gene regulation to a new level.