Dosage compensation (DC) in Drosophila equalizes X chromosome-linked gene expression via the male-specific lethal complex, which acetylates histone 4 lysine-16 on the male X chromosome. How DC is established during early embryogenesis remains obscure, largely due to the difficulty of obtaining sufficient sex-specific material at early embryonic stages. Here, we developed a high-throughput embryo sorting strategy and combined newly synthesized RNA-sequencing and RNA polymerase II (Pol II) profiling to investigate the mechanisms underlying the onset of DC in vivo. We found that changes in transcription initiation occur before detectable differences in transcriptional pausing or elongation and represent the primary determinant of enhanced mRNA output during DC establishment. It had hitherto been unclear whether DC establishment and maintenance exerted similar or distinct effects on transcriptional kinetics. Extending genome-wide profiling to S2 cells, we uncovered Pol II initiation as a general principle underlying both inception and perpetuation of DC-driven transcriptional up-regulation.

Influenza virus mRNAs are stable and competent for nuclear export and translation because they receive a 5' cap(1) structure in a process called cap snatching1. During cap snatching, the viral RNA-dependent RNA polymerase (FluPol) binds to host RNA polymerase II (Pol II) and the emerging transcript2,3. The FluPol endonuclease then cleaves a capped RNA fragment that subsequently acts as a primer for the transcription of viral genes4,5. Here we present the cryogenic electron microscopy structure of FluPol bound to a transcribing Pol II in complex with the elongation factor DSIF in the pre-cleavage state. The structure shows that FluPol directly interacts with both Pol II and DSIF, positioning the FluPol endonuclease domain near the RNA exit channel of Pol II. These interactions are important for the endonuclease activity of FluPol and FluPol activity in cells. A second structure, trapped after cap snatching, shows that the cleaved capped RNA rearranges within FluPol, directing the capped RNA 3' end toward the FluPol polymerase active site for viral transcription initiation. Together, our results provide the molecular mechanisms of co-transcriptional cap snatching by FluPol.

Transcription initiation by RNA polymerase II (RNA Pol II) occurs next to a +1 nucleosome, which is positioned downstream of the transcription start site (TSS). The +1 nucleosome influences pre-initiation complex (PIC) assembly and RNA Pol II pausing, but its function in transcription initiation and the transition to elongation remains unclear. Here, we investigate the transcription initiation-elongation transition in vitro using DNA templates containing a +1 nucleosome and present cryo-electron microscopy (cryo-EM) structures of five intermediate states. First, after PIC assembly, ATP binding to TFIIH enables the +1 nucleosome to evict TFIID from the PIC. Following DNA opening, the +1 nucleosome stimulates TFIIH translocase activity and initial RNA synthesis. Finally, after DNA bubble rewinding, the +1 nucleosome removes TFIIH from the early elongation complex for promoter escape. Our findings show that the +1 nucleosome not only acts passively in PIC assembly and RNA Pol II pausing but rather has active functions during the initiation-elongation transition of transcription.

RNA polymerase II (Pol II) is a 12-subunit enzyme crucial for gene transcription in the nucleus. However, its assembly in the cytoplasm, nuclear import, and nuclear function of assembly factors remain poorly understood. Here, we isolated Pol II from the cytoplasmic fraction of human cells (cfPol II) and determined its cryo-EM structure. The structure reveals that Pol II is fully assembled in the cytoplasm before nuclear import. We also found that Gdown1 binds Pol II through three distinct regions, indicating it may stabilize Pol II assembly intermediates. Notably, Gdown1 binding precludes the association of essential transcription factors IIB and IIF, rendering cfPol II inactive in promoter-dependent transcription in vitro . Our results provide a basis for Gdown1-dependent global transcription repression and suggest a model for the role of Gdown1 in Pol II assembly, import, and transcription regulation.

Transcription factors (TFs) can access nucleosomes via five distinct modes: gyre-spanning, periodic-binding, dyad-binding, and end-binding modes as well as an oriented binding mode, where the TF binding motif shows orientational preference relative to the nucleosome. Here, we report the first structure of an oriented TF:nucleosome complex, where two ELF2 proteins bind to a double motif located at superhelical location +4, unwinding four helical turns of DNA from the nucleosome. We further show that unlike previously described pioneer factors, ELF2 is able to occupy all of its unmethylated, high-affinity double motifs in vivo. Motifs of ELF2 and another oriented nucleosome binder, YY1, are highly enriched downstream of transcription start sites (TSSs) of highly expressed genes, with the motifs oriented in such a way that the TSS becomes accessible upon TF binding. Our results suggest that oriented binding may be generally important for high transcriptional activity.

SUMMARY Transcription initiation by RNA polymerase II (Pol II) occurs next to a +1 nucleosome that is positioned downstream of the transcription start site (TSS). The +1 nucleosome has been shown to influence the pre-initiation complex (PIC) assembly and Pol II pausing, but it is unclear whether and how it functions in transcription initiation and the transition to elongation. Here, we investigate the transcription initiation-elongation transition in vitro using DNA templates containing a +1 nucleosome, and we present cryo-EM structures of five intermediate states. First, after PIC assembly, the +1 nucleosome evicts TFIID from the PIC upon binding of ATP to TFIIH. Second, after DNA opening, the +1 nucleosome stimulates TFIIH translocase activity and initial RNA synthesis. Finally, after DNA bubble rewinding, the +1 nucleosome removes TFIIH from the early elongation complex for promoter escape. Our findings show that the +1 nucleosome not only acts as a passive border for PIC assembly and a passive barrier for Pol II pausing, but rather has active functions during the initiation-elongation transition of transcription.

Targeting the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 with small molecules is a promising therapeutic strategy against COVID-19, but potent and safe inhibitors are lacking. HeE1-2Tyr, a nonnucleoside inhibitor of Dengue virus RdRp, was also shown to inhibit SARS-CoV-2 RdRp in vitro and to have antiviral activity in cells, but the underlying mechanism remains unclear. Here, we elucidate the molecular mechanism of HeE1-2Tyr-mediated SARS-CoV-2 RdRp inhibition. Biochemical assays confirm that HeE1-2Tyr inhibits RdRp with an IC50 of 5 µM and show that it competes with RNA binding to RdRp in vitro. Structural analysis using cryo-EM reveals that a stack of three HeE1-2Tyr molecules binds to the RNA binding site of RdRp. The identification of the conserved HeE1-2Tyr binding site and its intriguing inhibition mechanism of three stacked molecules that outcompete RNA may facilitate further development of pan-corona nonnucleoside inhibitors.

RNA polymerase II (Pol II) facilitates co-transcriptional splicing by recruiting the U1 small nuclear ribonucleoprotein particle (U1 snRNP) to the nascent transcripts. Here, we report the cryo-electron microscopy structure of a transcribing Pol II-U1 snRNP complex with elongation factors DSIF and SPT6. Furthermore, our biochemical analysis revealed that the phosphorylated Pol II carboxyl-terminal domain and SPT6 interact directly with U1 snRNP proteins, facilitating its recruitment to the elongation complex. This multivalent interaction allows efficient spliceosome assembly and ensures transcription processivity. ### Competing Interest Statement The authors have declared no competing interest.

UV (ultra-violet) crosslinking with mass spectrometry (XL-MS) has been established for identifying RNA- and DNA-binding proteins along with their domains and amino acids involved. Here, we explore chemical XL-MS for RNA-protein, DNA-protein, and nucleotide-protein complexes in vitro and in vivo. We introduce a specialized nucleotide-protein-crosslink search engine, NuXL, for robust and fast identification of such crosslinks at amino acid resolution. Chemical XL-MS complements UV XL-MS by generating different crosslink species, increasing crosslinked protein yields in vivo almost four-fold and thus it expands the structural information accessible via XL-MS. Our workflow facilitates integrative structural modelling of nucleic acid-protein complexes and adds spatial information to the described RNA-binding properties of enzymes, for which crosslinking sites are often observed close to their cofactor-binding domains. In vivo UV and chemical XL-MS data from E. coli cells analysed by NuXL establish a comprehensive nucleic acid-protein crosslink inventory with crosslink sites at amino acid level for more than 1500 proteins. Our new workflow combined with the dedicated NuXL search engine identified RNA crosslinks that cover most RNA-binding proteins, with DNA and RNA crosslinks detected in transcriptional repressors and activators.

The protein IWS1 (Interacts with SPT6 1) is implicated in transcription-associated processes, but a direct role in RNA polymerase (Pol) II function is unknown. Here, we use multi-omics kinetic analysis after rapid depletion of IWS1 in human cells to show that loss of IWS1 results in a global decrease of RNA synthesis and a global reduction in Pol II elongation velocity. We then resolve the cryo-EM structure of the activated Pol II elongation complex with bound IWS1 and elongation factor ELOF1 and show that IWS1 acts as a scaffold and positions downstream DNA within the cleft of Pol II. In vitro assays show that the disordered C-terminal region of IWS1 that contacts the cleft of Pol II is responsible for stimulation of Pol II activity and is aided by ELOF1. Finally, we find that the defect in transcription upon IWS1 depletion leads to a decrease of histone H3 tri-methylation at residue lysine-36 (H3K36me3), but that this secondary effect is an indirect function of IWS1. In summary, our structure-function analysis establishes IWS1 as a Pol II-associated elongation factor that acts globally to stimulate Pol II elongation velocity and ensure proper co-transcriptional histone methylation.

In eukaryotic cells, splicing occurs predominantly co-transcriptionally, enhancing splicing efficiency and fidelity while introducing an additional layer of regulation over gene expression. RNA polymerase II (Pol II) facilitates co-transcriptional splicing by recruiting the U1 small nuclear ribonucleoprotein particle (U1 snRNP) to the nascent transcripts. Here, we report the cryo-electron microscopy structure of a transcribing Pol II-U1 snRNP complex with elongation factors DSIF and SPT6. In addition, our biochemical analysis reveals that the phosphorylated Pol II carboxyl-terminal domain and SPT6 interact directly with U1 snRNP proteins, facilitating its recruitment to the elongation complex. This multivalent interaction between U1 snRNP and the transcription elongation complex may both allow efficient spliceosome assembly and ensure transcription processivity.

Metazoan gene transcription by RNA polymerase II (Pol II) is regulated in the promoter-proximal region. Pol II can undergo termination in the promoter-proximal region but whether this can contribute to transcription regulation in cells remains unclear. Here we extend our previous multiomics analysis to quantify changes in transcription kinetics during a human cell type transition event. We observe that upregulation of transcription involves an increase in initiation frequency and, at a set of genes, a decrease in promoter-proximal termination. In turn, downregulation of transcription involves a decrease in initiation frequency and an increase in promoter-proximal termination. Thus, promoter-proximal termination of Pol II contributes to the regulation of human gene transcription.

H3K36me3 is a hallmark of actively and recently transcribed genes and contributes to cellular memory and identity. The deposition of H3K36me3 occurs co-transcriptionally when the methyltransferase SETD2 associates with RNA polymerase II. Here we present three cryo-EM structures of SETD2 bound to RNA polymerase II elongation complexes at different states of nucleosome passage. Together with functional probing, our results suggest a 3-step mechanism of transcription-coupled H3K36me3 deposition. First, binding to the elongation factor SPT6 tethers the catalytic SET domain in proximity to the upstream DNA. Second, RNA polymerase II nucleosome passage leads to the transfer of a hexasome from downstream to upstream, poised for methylation. Finally, continued transcription leads to upstream nucleosome reassembly, partial dissociation of the histone chaperone FACT and sequential methylation of both H3 tails, completing H3K36me3 deposition of an upstream nucleosome after RNA polymerase II passage.

RNA polymerase II (Pol II) transcription is regulated by many elongation factors. Among these factors, TFIIF, PAF-RTF1, ELL and Elongin stimulate mRNA chain elongation by Pol II. Cryo-EM structures of Pol II complexes with these elongation factors now reveal some general principles on how elongation factors bind Pol II and how they stimulate transcription. All four elongation factors contact Pol II at domains external 2 and protrusion, whereas TFIIF and ELL additionally bind the Pol II lobe. All factors apparently stabilize cleft-flanking elements, whereas RTF1 and Elongin additionally approach the active site with a latch element and may influence catalysis or translocation. Due to the shared binding sites on Pol II, factor binding is mutually exclusive, and thus it remains to be studied what determines which elongation factors bind at a certain gene and under which condition.

Eukaryotic genomes are organized into chromatin domains. The molecular mechanisms driving the formation of these domains are difficult to dissect in vivo and remain poorly understood. Here we reconstitute Saccharomyces cerevisiae chromatin in vitro and determine its 3D organization at subnucleosome resolution by micrococcal nuclease-based chromosome conformation capture and molecular dynamics simulations. We show that regularly spaced and phased nucleosome arrays form chromatin domains in vitro that resemble domains in vivo. This demonstrates that neither loop extrusion nor transcription is required for basic domain formation in yeast. In addition, we find that the boundaries of reconstituted domains correspond to nucleosome-free regions and that insulation strength scales with their width. Finally, we show that domain compaction depends on nucleosome linker length, with longer linkers forming more compact structures. Together, our results demonstrate that regular nucleosome positioning is important for the formation of chromatin domains and provide a proof-of-principle for bottom-up 3D genome studies.

The transition from transcription initiation to elongation is highly regulated in human cells but remains incompletely understood at the structural level. In particular, it is unclear how interactions between RNA polymerase II (RNA Pol II) and initiation factors are broken to enable promoter escape. Here, we reconstitute RNA Pol II promoter escape in vitro and determine high-resolution structures of initially transcribing complexes containing 8-, 10-, and 12-nt ordered RNAs and two elongation complexes containing 14-nt RNAs. We suggest that promoter escape occurs in three major steps. First, the growing RNA displaces the B-reader element of the initiation factor TFIIB without evicting TFIIB. Second, the rewinding of the transcription bubble coincides with the eviction of TFIIA, TFIIB, and TBP. Third, the binding of DSIF and NELF facilitates TFIIE and TFIIH dissociation, establishing the paused elongation complex. This three-step model for promoter escape fills a gap in our understanding of the initiation-elongation transition of RNA Pol II transcription.

To maintain the nucleosome organization of transcribed genes, ATP-dependent chromatin remodelers collaborate with histone chaperones. Here, we show that at the 5' ends of yeast genes, RNA polymerase II (RNAPII) generates hexasomes that occur directly adjacent to nucleosomes. The resulting hexasome-nucleosome complexes are then resolved by Chd1. We present two cryoelectron microscopy (cryo-EM) structures of Chd1 bound to a hexasome-nucleosome complex before and after restoration of the missing inner H2A/ H2B dimer by FACT. Chd1 uniquely interacts with the complex, positioning its ATPase domain to shift the hexasome away from the nucleosome. In the absence of the inner H2A/H2B dimer, its DNA-binding domain (DBD) packs against the ATPase domain, suggesting an inhibited state. Restoration of the dimer by FACT triggers a rearrangement that displaces the DBD and stimulates Chd1 remodeling. Our results demonstrate how chromatin remodelers interact with a complex nucleosome assembly and suggest how Chd1 and FACT jointly support transcription by RNAPII.

The mechanisms underlying the initiation and elongation of RNA polymerase II (Pol II) transcription are well-studied, whereas termination remains poorly understood. Here we analyze the mechanism of polyadenylation-independent Pol II termination mediated by the yeast Sen1 helicase. Cryo-electron microscopy structures of two pretermination intermediates show that Sen1 binds to Pol II and uses its adenosine triphosphatase activity to pull on exiting RNA in the 5 ' direction. This is predicted to push Pol II forward, induce an unstable hypertranslocated state and destabilize the transcription bubble, thereby facilitating termination. This mechanism of transcription termination may be widely used because it is conceptually conserved in the bacterial transcription system. In this work, the authors report how the termination factor Sen1 interacts with an elongating RNA polymerase II (Pol II) and its nascent transcript to perform termination. Comparison of two pretermination states of Pol II supports a hypertranslocation model of termination.