| Transcription is a genetic transmission process in which double-strand DNA guides the synthesis of single-strand RNA molecules.The largest subunit of eukaryotic RNA polymerase Ⅱ(Pol Ⅱ),Rpb1,contains a peculiar C-terminus domain(CTD),composed of highly conserved heptapeptide repeats(YSPTSPS)with extremely flexible structure and dynamic post-translational modifications.During transcriptional elongation,the hyperphosphorylated CTD performs as a binding platform for multiple elongation factors,epigenetic factors and RNA processing factors.Histone H3 lysine 36(H3K36me)methyltransferase Set2/SETD2 is one of them,recognizing CTD Ser2/Ser5 phosphorylation sites,co-transcriptionally mediating H3K36me3 modification.H3K36me3 site is a hallmark of actively transcribing genes,involved in pathways such as transcriptional fidelity maintenance and pre-mRNA splicing.The structure of Set2-Pol Ⅱ holoenzyme remains elusive,limiting to the interpretation of co-transcriptional regulation of elongation efficiency by Set2/SETD2.We successfully assembled the Set2-Pol Ⅱ complex in vitro by biochemical methods and verified that purified Set2 possesses the activity of methylating histone and nucleosome substrates.It is consistent with previous reported data that the binding of Set2 to Pol Ⅱ mainly depends on CTD Ser2 phosphorylation site and supplements by Ser5 phosphorylation site.Combining with negative staining random conical tilt(RCT)reconstruction and cryo-EM single particle 3D reconstruction,we preliminarily analyzed the structure of full-length Set2-Pol Ⅱ complex.Two interaction manners of Set2-Pol Ⅱ were found in both reconstruction ways.The binding regions include the Rpb1 clamp region,jaw region and linker-CTD region.Results of chemical crosslinking and mass spectrometry(XL-MS)indicated the intermolecular crosslinking lysine pairs were between SET domain of Set2 to clamp domain of Rpb1 and SRI(Set2-Rpb1 interaction)domain of Set2 to jaw region of Rpb1.Our 3D reconstruction and XL-MS results consistently displayed that SET-clamp and SRI-CTD were the main interaction mode between Set2 and Pol Ⅱ.Using Ni+-NTA nanogolds,we labeled the C-terminal position of CTD and verified this conclusion.The highly phosphorylated CTD during elongation was partially localized to Pol Ⅱmain body,and two sets of localization patterns was consistent with the binding pattern of Set2-Pol Ⅱ found in 3D reconstruction.In addition,we resolved the preliminary structure of Set2-Pol Ⅱ-NCPH3K36M(nucleosome core particle,NCP)complex under negative staining conditions.Pol Ⅱassociated to NCP at a fixed orientation:the clamp and jaw/lobe regions on both sides of Pol Ⅱ were probably responsible for the interaction with super-helical DNA on the surface of NCP,consistent with recently published high-resolution structural observations of Pol Ⅱ-NCP complex.Set2 mainly associated to NCP.Owing to the limitation of biochemical sample preparation,we did not observe a clear structure of Set2-Pol Ⅱ-NCP ternary complex.In conclusion,with the assistant of XL-MS and labeling by nanogold,the preliminary structure of Set2-Pol Ⅱ complex demonstrated thatSet2-Pol Ⅱ interact mutually by the SET-clamp and SRI-CTD axes.We proposed a mechanistic model that Pol Ⅱ co-transcriptionally regulates the methyltxansferase activity of Set2 through dynamic conformational changes of clamp region.This study is practicable to interpretation of Set2-Pol Ⅱ interaction mode and provide a basis for further structural analysis. |
