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Teaser, summary, work performed and final results

Periodic Reporting for period 3 - DECOR (Dynamic assembly and exchange of RNA polymerase II CTD factors)

Teaser

The C-terminal domain (CTD) of the RNA polymerase II (RNAPII) largest subunit coordinates co-transcriptional processing and it is decorated by many processing factors throughout the transcription cycle. To determine how these accessory factors assemble and exchange on the CTD...

Summary

The C-terminal domain (CTD) of the RNA polymerase II (RNAPII) largest subunit coordinates co-transcriptional processing and it is decorated by many processing factors throughout the transcription cycle. To determine how these accessory factors assemble and exchange on the CTD of RNAPII has remained a major challenge. In this project, we aim to unravel the structural and mechanistic bases for the dynamic assembly of RNAPII CTD with its transcription and processing factors.
The architecture and dynamics of CTDsome are not only important for a general understanding of transcription regulation and co-transcriptional processing, but also have implications for the design of novel therapies. For example, viral RNA polymerase of influenza binds the CTD in order to replicate viral RNAs. The detailed knowledge of interactions in the CTDsome will be critical for the future development of new anti-viral agents.
Our study will answer the long-standing questions of how the overall CTD structure is modulated on binding to processing factors, and whether these factors cross-talk and compete with each other.

Work performed

In the first period for finantial reporting of the project we reached several important milestones: (i) we prepared and biochemically characterized the studied proteins and their fragments, (ii) we prepared stable protein-peptide complexes and performed their characterization, (iii) we carried out NMR data acquisition and resonance assignments of the studied complexes, (iv) we performed SAXS and X-ray data measurement and data analyses (v) We determined the first structures of the studied complexes, (vi) we started with functional analyses of the studied systems. Altogether, this is in accord with the proposed plan and it will enable us to probe the structural basis of the CTD code and CTDsome assembly in the next years of the project.
We wish to highlight that the first structural and biochemical data from this project were just accepted for publication in EMBO reports. In this paper in press, we reported the structural and functional characterization of Thr4 phosphorylated CTD-- Rtt103 complex. Our data reveal a direct recognition of phosphorylated Thr4 by Rtt103p, the phosphorylation mark previously thought to be incompatible with recruitment of CTD reader factors. Based on our data, we proposed that the CTD code of RNAP II is degenerated, a feature which is similar to the codon degeneracy.

Since the beginning of the project until the first scientific report we aimed to unravel the structural and mechanistic bases for the dynamic assembly of C-terminal domain (CTD) of the RNA polymerase II (RNAPII) with its processing factors. During transcription, many of processing factors associate with RNAPII weakly and transiently, and the association is dictated by different phosphorylation patterns and conformational changes of the CTD. To determine how these accessory factors decorate the CTD—how they assemble and exchange on the CTD—we use a combination of NMR, X-ray crystallography, small-angle X-ray scattering, and recently also cryo-EM approaches, along with biochemical tools. The project DECOR has four aims which all together should provide a model for the RNAP II CTDsome assembly and dynamics. The work resulted in publication of two papers and two other manuscript are under review at the time of the submission of this report. Three additional manuscript are currently in preparation.
Aim 1. Determine 3D structures of protein factors bound to the modified CTD.
We determined structures of several protein factors bound to the RNAP II CTD carrying specific phosphorylations and characterized their binding affinities. This enables us to decode how CTD phosphorylation patterns stimulate or prevent binding of a given processing factor. This structural work is important to decipher the basic rules that govern the readout of the CTD code of RNA polymerase II by RNA processing factors. Generally, we revealed two protein families of phospho-CTD readers (protein domains reading phosphomarks of the CTD). One family is very sensitive to specific phosphorylation in the CTD -- as certain phosphomarks stimulate their binding whereas others prevent their binding. Another family of the phospho-CTD readers can be recruited to the CTD equally-well for different CTD phosphorylations and no combination of phosphomarks can prevent their binding. Our data also suggest that the CTD code of RNAP II is degenerated. In other words, the recruitment of a single CTD binding factor may be coded by more than one letter of the CTD code.
We solved structures of transcription and processing factors that read the phospho-CTD marks occurring at the beginning, in the middle, and at the end of transcription cycle. To name an example, we solved the structure and rationalized the specificity of human RPRD2 (manuscript in preparation) and yeast Rtt103 (Jasnovidova et al. PNAS 2017) which recognize the “late” phospho-CTD marks (CTD phosphorylated on serine 2). We also found that the affinity further increases if serine 7 is also phosphorylated, whereas serine 5

Final results

The CTDsome assembly/disassembly is encoded by the so called CTD code. This concept which was proposed almost a decade ago considers that the association of specific factors with the CTD is dictated by different post-translational modification patterns and conformational changes in the CTD. Within this project, we found that the CTD code of RNAP II is degenerated, that is the recruitment of a single CTD binding factor may be coded for by more than one letter of the CTD code. This is conceptually new consideration in the field of transcription regulation and has a number of practical consequences. For example, it explains how CTD binding factors can be recruited to the poorly conserved heptad repeats, which are frequently present in all eukaryotes It also explains how the CTD-binding factors can tolerate some errors or imperfections in phosphorylation of the CTD.

Website & more info

More info: https://www.ceitec.eu/decor-dynamic-assembly-and-exchange-of-rna-polymerase-ii-ctd-factors/t2960.