Structural and functional characterization of the interaction between the influenza A virus RNA polymerase and the CTD of host RNA polymerase II.

Influenza A viruses, causing seasonal epidemics and occasional pandemics, rely on interactions with host proteins for their RNA genome transcription and replication. The viral RNA polymerase utilizes host RNA polymerase II (Pol II) and interacts with the serine 5 phosphorylated (pS5) C-terminal domain (CTD) of Pol II to initiate transcription. Our study, using single-particle electron cryomicroscopy (cryo-EM), reveals the structure of the 1918 pandemic influenza A virus polymerase bound to a synthetic pS5 CTD peptide composed of four heptad repeats mimicking the 52 heptad repeat mammalian Pol II CTD. The structure shows that the CTD peptide binds at the C-terminal domain of the PA viral polymerase subunit (PA-C) and reveals a previously unobserved position of the 627 domain of the PB2 subunit near the CTD. We identify crucial residues of the CTD peptide that mediate interactions with positively charged cavities on PA-C, explaining the preference of the viral polymerase for pS5 CTD. Functional analysis of mutants targeting the CTD-binding site within PA-C reveals reduced transcriptional function or defects in replication, highlighting the multifunctional role of PA-C in viral RNA synthesis. Our study provides insights into the structural and functional aspects of the influenza virus polymerase-host Pol II interaction and identifies a target for antiviral development.IMPORTANCEUnderstanding the intricate interactions between influenza A viruses and host proteins is crucial for developing targeted antiviral strategies. This study employs advanced imaging techniques to uncover the structural nuances of the 1918 pandemic influenza A virus polymerase bound to a specific host protein, shedding light on the vital process of viral RNA synthesis. The study identifies key amino acid residues in the influenza polymerase involved in binding host polymerase II (Pol II) and highlights their role in both viral transcription and genome replication. These findings not only deepen our understanding of the influenza virus life cycle but also pinpoint a potential target for antiviral development. By elucidating the structural and functional aspects of the influenza virus polymerase-host Pol II interaction, this research provides a foundation for designing interventions to disrupt viral replication and transcription, offering promising avenues for future antiviral therapies.

Chemoenzymatic Synthesis of 4,5-Dihydroxyisoleucine Fragment of α-Amanitin.

The ability of α-amanitin to potently inhibit RNA polymerase II (RNAP II) has elicited further research into its use as a novel payload for antibody-drug conjugates. Despite this promise, the de novo synthesis of α-amanitin is still a major challenge as it possesses an unusual bicyclic octapeptide structure that contains several oxidized amino acids, most notably 4,5-dihydroxy-l-isoleucine. Here, we report a concise chemoenzymatic synthesis of this key amino acid residue, which features two regioselective and diastereoselective enzymatic C-H oxidations on l-isoleucine.

Structural basis of Integrator-dependent RNA polymerase II termination.

The Integrator complex can terminate RNA polymerase II (Pol II) in the promoter-proximal region of genes. Previous work has shed light on how Integrator binds to the paused elongation complex consisting of Pol II, the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF) and how it cleaves the nascent RNA transcript1, but has not explained how Integrator removes Pol II from the DNA template. Here we present three cryo-electron microscopy structures of the complete Integrator-PP2A complex in different functional states. The structure of the pre-termination complex reveals a previously unresolved, scorpion-tail-shaped INTS10-INTS13-INTS14-INTS15 module that may use its 'sting' to open the DSIF DNA clamp and facilitate termination. The structure of the post-termination complex shows that the previously unresolved subunit INTS3 and associated sensor of single-stranded DNA complex (SOSS) factors prevent Pol II rebinding to Integrator after termination. The structure of the free Integrator-PP2A complex in an inactive closed conformation2 reveals that INTS6 blocks the PP2A phosphatase active site. These results lead to a model for how Integrator terminates Pol II transcription in three steps that involve major rearrangements.

Multiphosphorylation-Dependent Recognition of Anti-pS2 Antibodies against RNA Polymerase II C-Terminal Domain Revealed by Chemical Synthesis.

Phosphorylation is a major constituent of the CTD code, which describes the set of post-translational modifications on 52 repeats of a YSPTSPS consensus heptad that orchestrates the binding of regulatory proteins to the C-terminal domain (CTD) of RNA polymerase II. Phospho-specific antibodies are used to detect CTD phosphorylation patterns. However, their recognition repertoire is underexplored due to limitations in the synthesis of long multiphosphorylated peptides. Herein, we describe the development of a synthesis strategy that provides access to multiphosphorylated CTD peptides in high purity without HPLC purification for immobilization onto microtiter plates. Native chemical ligation was used to assemble 12 heptad repeats in various phosphoforms. The synthesis of >60 CTD peptides, 48-90 amino acids in length and containing up to 6 phosphosites, enabled a detailed and rapid analysis of the binding characteristics of different anti-pSer2 antibodies. The three antibodies tested showed positional selectivity with marked differences in the affinity of the antibodies for pSer2-containing peptides. Furthermore, the length of the phosphopeptides allowed a systematic analysis of the multivalent chelate-type interactions. The absence of multivalency-induced binding enhancements is probably due to the high flexibility of the CTD scaffold. The effect of clustered phosphorylation proved to be more complex. Recognition of pSer2 by anti-pSer2-antibodies can be prevented and, perhaps surprisingly, enhanced by the phosphorylation of "bystander" amino acids in the vicinity. The results have relevance for functional analysis of the CTD in cell biological experiments.

Structural basis of exoribonuclease-mediated mRNA transcription termination.

Efficient termination is required for robust gene transcription. Eukaryotic organisms use a conserved exoribonuclease-mediated mechanism to terminate the mRNA transcription by RNA polymerase II (Pol II)1-5. Here we report two cryogenic electron microscopy structures of Saccharomyces cerevisiae Pol II pre-termination transcription complexes bound to the 5'-to-3' exoribonuclease Rat1 and its partner Rai1. Our structures show that Rat1 displaces the elongation factor Spt5 to dock at the Pol II stalk domain. Rat1 shields the RNA exit channel of Pol II, guides the nascent RNA towards its active centre and stacks three nucleotides at the 5' terminus of the nascent RNA. The structures further show that Rat1 rotates towards Pol II as it shortens RNA. Our results provide the structural mechanism for the Rat1-mediated termination of mRNA transcription by Pol II in yeast and the exoribonuclease-mediated termination of mRNA transcription in other eukaryotes.

Mechanisms and Functions of the RNA Polymerase II General Transcription Machinery during the Transcription Cycle.

Central to the development and survival of all organisms is the regulation of gene expression, which begins with the process of transcription catalyzed by RNA polymerases. During transcription of protein-coding genes, the general transcription factors (GTFs) work alongside RNA polymerase II (Pol II) to assemble the preinitiation complex at the transcription start site, open the promoter DNA, initiate synthesis of the nascent messenger RNA, transition to productive elongation, and ultimately terminate transcription. Through these different stages of transcription, Pol II is dynamically phosphorylated at the C-terminal tail of its largest subunit, serving as a control mechanism for Pol II elongation and a signaling/binding platform for co-transcriptional factors. The large number of core protein factors participating in the fundamental steps of transcription add dense layers of regulation that contribute to the complexity of temporal and spatial control of gene expression within any given cell type. The Pol II transcription system is highly conserved across different levels of eukaryotes; however, most of the information here will focus on the human Pol II system. This review walks through various stages of transcription, from preinitiation complex assembly to termination, highlighting the functions and mechanisms of the core machinery that participates in each stage.

RNA polymerase II elongation factors use conserved regulatory mechanisms.

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.

The Ser7 of RNA Pol II-CTD influences the recruitment of Cdc73 for mRNA transcription.

The carboxyl terminal domain of the largest subunit of eukaryotic RNA polymerase II (RNAPII) consists of highly conserved tandem repeats of Tyr1Ser2Pro3Thr4Ser5Pro6Ser7, referred as CTD. The CTD undergoes posttranslational modifications where the interplay of kinases imparts specific CTD phosphorylations, recognized by regulatory proteins that help in the mRNA transcription. Here, the Ser5 phosphorylation (Ser5P) remains high during the transcription initiation, followed by the Ser2P which peaks towards the termination and the Ser7P remains high throughout the transcription process. The Paf1 elongation complex (Paf1C) through its Cdc73 subunit is recruited to the phosphorylated CTD and play active role during different stages of mRNA transcription. We show that the CTD binding domain of Cdc73 is an independent folding unit which interacts with the hyper phosphorylated CTD. The 500 ns MD simulation studies further identified the binding interface and the pattern of CTD phosphorylation involved in the interaction with Cdc73. The possible key residues were mutated and the subsequent pull down analysis suggests that the phosphorylated Ser2, Ser5 and Ser7 of the tandem CTD heptads interact respectively with Arg310, Arg268 and Arg300 of Cdc73. Our finding provides new insight for Cdc73 function during mRNA transcription.

Structural visualization of transcription initiation in action.

Transcription initiation is a complex process, and its mechanism is incompletely understood. We determined the structures of de novo transcribing complexes TC2 to TC17 with RNA polymerase II halted on G-less promoters when nascent RNAs reach 2 to 17 nucleotides in length, respectively. Connecting these structures generated a movie and a working model. As initially synthesized RNA grows, general transcription factors (GTFs) remain bound to the promoter and the transcription bubble expands. Nucleoside triphosphate (NTP)-driven RNA-DNA translocation and template-strand accumulation in a nearly sealed channel may promote the transition from initially transcribing complexes (ITCs) (TC2 to TC9) to early elongation complexes (EECs) (TC10 to TC17). Our study shows dynamic processes of transcription initiation and reveals why ITCs require GTFs and bubble expansion for initial RNA synthesis, whereas EECs need GTF dissociation from the promoter and bubble collapse for promoter escape.

The Saccharomyces cerevisiae SR protein Npl3 interacts with hyperphosphorylated CTD of RNA Polymerase II.

The catalytic subunit of RNA Polymerase II contains a highly conserved carboxy terminal domain (CTD) composed of multiple tandem heptad sequence Tyr1Ser2Pro3Thr4Ser5Pro6Ser7. The non-proline residues in CTD undergo posttranslational modifications, with Ser5 phosphorylation (Ser5P) predominating at the start of the transcription cycle and Ser2P at the end, while other phosphorylation levels are high all throughout. The differentially phosphorylated CTD is recognized by regulatory proteins, helpful during mRNA transcription and export. One such protein Npl3 is composed of two RNA binding domains and a C-terminus RGG/SR domain. The Ser411 of Npl3 is reported to make direct contact with Ser2P of CTD for its recruitment and function, while the Npl3 lacking of C-terminal 25 amino acids (Npl3Δ389-414) showed no apparent defects in mRNA synthesis. Here, we report that the RNA binding domains of Npl3 are separate folding units and interact also with the CTD. The interaction between Npl3 and CTD appears to involve not just Ser2P, but also the Ser5P and Ser7P. The Arg126 of the first RNA binding domain interacts with Ser2P whereas the Arg235 of the second RNA binding domain interacts with either Ser7P or Ser5P of another heptad. The finding provides new insight of Npl3 function for mRNA transcription.

Complex Conformational Space of the RNA Polymerase II C-Terminal Domain upon Phosphorylation.

Intrinsically disordered proteins (IDPs) have been closely studied during the past decade due to their importance in many biological processes. The disordered nature of this group of proteins makes it difficult to observe its full span of the conformational space using either experimental or computational studies. In this article, we explored the conformational space of the C-terminal domain (CTD) of RNA polymerase II (Pol II), which is also an intrinsically disordered low complexity domain, using enhanced sampling methods. We provided a detailed conformational analysis of model systems of CTD with different lengths; first with the last 44 residues of the human CTD sequence and finally the CTD model with 2-heptapeptide repeating units. We then investigated the effects of phosphorylation on CTD conformations by performing simulations at different phosphorylated states. We obtained broad conformational spaces in nonphosphorylated CTD models, and phosphorylation has complex effects on the conformations of the CTD. These complex effects depend on the length of the CTD, spacing between the multiple phosphorylation sites, ion coordination, and interactions with the nearby residues.

RNA Polymerase II evolution and adaptations: Insights from Plasmodium and other parasitic protists.

The C-terminal domain (CTD) of RNA polymerase II plays a crucial role in regulating transcription dynamics in eukaryotes. The phosphorylation of serine residues within the CTD controls transcription initiation, elongation, and termination. While the CTD is highly conserved across eukaryotes, lower eukaryotes like protists, including Plasmodium, exhibit some differences. In this study, we performed a comparative analysis of CTD in eukaryotic systems to understand why the parasites evolved in this particular manner. The Plasmodium falciparum RPB1 is exceptionally large and feature a gap between the first and second heptad repeats, resulting in fifteen canonical heptad repeats excluding the initial repeat. Analysis of this intervening sequence revealed sub motifs of heptads where two serine residues occupy the first and fourth positions (S1X2X3S4). These motifs lie in the intrinsically disordered region of RPB1, a characteristic feature of the CTD. Interestingly, the S1X2X3S4 sub-motif was also observed in early-divergingeukaryotes like Leishmania major, which lack canonical heptad repeats. Furthermore, eukaryotes across the phylogenetic tree revealed a sigmoid pattern of increasing serine frequency in the CTD, indicating that serine enrichment is a significant step in the evolution of heptad-rich RPB1. Based on these observations and analysis, we proposed an evolutionary model for RNA Polymerase II CTD, encompassing organisms previously deemed exceptions, notably Plasmodium species. Thus, our study provides novel insights into the evolution of the CTD and will prompt further investigations into the differences exhibited by Plasmodium RNA Pol II and determine if they confer a survival advantage to the parasite.

Structural basis for evolutionarily conserved interactions between TFIIS and Paf1C.

The elongation factor TFIIS interacts with Paf1C complex to facilitate processive transcription by Pol II. We here determined the crystal structure of the trypanosoma TFIIS LW domain in a complex with the LFG motif of Leo1, as well as the structures of apo-form TFIIS LW domains from trypanosoma, yeast and human. We revealed that all three TFIIS LW domains possess a conserved hydrophobic core that mediates their interactions with Leo1. Intriguingly, the structural study revealed that trypanosoma Leo1 binding induces the TFIIS LW domain to undergo a conformational change reflected in the length and orientation of α6 helix that is absent in the yeast and human counterparts. These differences explain the higher binding affinity of the TFIIS LW domain interacting with Leo1 in trypanosoma than in yeast and human, and indicate species-specific variations in the interactions. Importantly, the interactions between the TFIIS LW domain and an LFG motif of Leo1 were found to be critical for TFIIS to anchor the entire Paf1C complex. Thus, in addition to revealing a detailed structural basis for the TFIIS-Paf1C interaction, our studies also shed light on the origin and evolution of the roles of TFIIS and Paf1C complex in regulation of transcription elongation.

Chromatin expansion microscopy reveals nanoscale organization of transcription and chromatin.

Nanoscale chromatin organization regulates gene expression. Although chromatin is notably reprogrammed during zygotic genome activation (ZGA), the organization of chromatin regulatory factors during this universal process remains unclear. In this work, we developed chromatin expansion microscopy (ChromExM) to visualize chromatin, transcription, and transcription factors in vivo. ChromExM of embryos during ZGA revealed how the pioneer factor Nanog interacts with nucleosomes and RNA polymerase II (Pol II), providing direct visualization of transcriptional elongation as string-like nanostructures. Blocking elongation led to more Pol II particles clustered around Nanog, with Pol II stalled at promoters and Nanog-bound enhancers. This led to a new model termed "kiss and kick", in which enhancer-promoter contacts are transient and released by transcriptional elongation. Our results demonstrate that ChromExM is broadly applicable to study nanoscale nuclear organization.

ERK2 stimulates MYC transcription by anchoring CDK9 to the MYC promoter in a kinase activity-independent manner.

The transcription factor MYC regulates cell proliferation, transformation, and survival in response to growth factor signaling that is mediated in part by the kinase activity of ERK2. Because ERK2 can also bind to DNA to modify gene expression, we investigated whether it more directly regulates MYC transcription. We identified ERK2 binding sites in the MYC promoter and detected ERK2 at the promoter in various serum-stimulated cell types. Expression of nuclear-localized ERK2 constructs in serum-starved cells revealed that ERK2 in the nucleus-regardless of its kinase activity-increased MYC mRNA expression and MYC protein abundance. ERK2 bound to the promoter through its amino-terminal insert domain and to the cyclin-dependent kinase CDK9 (which activates RNA polymerase II) through its carboxyl-terminal conserved docking domain. Both interactions were essential for ERK2-induced MYC expression, and depleting ERK impaired CDK9 occupancy and RNA polymerase II progression at the MYC promoter. Artificially tethering CDK9 to the MYC promoter by fusing it to the ERK2 insert domain was sufficient to stimulate MYC expression in serum-starved cells. Our findings demonstrate a role for ERK2 at the MYC promoter acting as a kinase-independent anchor for the recruitment of CDK9 to promote MYC expression.

Structure and phase separation of the C-terminal domain of RNA polymerase II.

The repetitive heptads in the C-terminal domain (CTD) of RPB1, the largest subunit of RNA Polymerase II (Pol II), play a critical role in the regulation of Pol II-based transcription. Recent findings on the structure of the CTD in the pre-initiation complex determined by cryo-EM and the novel phase separation properties of key transcription components offers an expanded mechanistic interpretation of the spatiotemporal distribution of Pol II during transcription. Current experimental evidence further suggests an exquisite balance between CTD's local structure and an array of multivalent interactions that drive phase separation of Pol II and thus shape its transcriptional activity.

Structural insights into human co-transcriptional capping.

Co-transcriptional capping of the nascent pre-mRNA 5' end prevents degradation of RNA polymerase (Pol) II transcripts and suppresses the innate immune response. Here, we provide mechanistic insights into the three major steps of human co-transcriptional pre-mRNA capping based on six different cryoelectron microscopy (cryo-EM) structures. The human mRNA capping enzyme, RNGTT, first docks to the Pol II stalk to position its triphosphatase domain near the RNA exit site. The capping enzyme then moves onto the Pol II surface, and its guanylyltransferase receives the pre-mRNA 5'-diphosphate end. Addition of a GMP moiety can occur when the RNA is ∼22 nt long, sufficient to reach the active site of the guanylyltransferase. For subsequent cap(1) methylation, the methyltransferase CMTR1 binds the Pol II stalk and can receive RNA after it is grown to ∼29 nt in length. The observed rearrangements of capping factors on the Pol II surface may be triggered by the completion of catalytic reaction steps and are accommodated by domain movements in the elongation factor DRB sensitivity-inducing factor (DSIF).

Association of peptidyl prolyl cis/trans isomerase Rrd1 with C terminal domain of RNA polymerase II.

The largest subunit of RNAPII extends as the conserved unstructured heptapeptide consensus repeats Y1S2P3T4S5P6S7 and their posttranslational modification, especially the phosphorylation state at Ser2, Ser5 and Ser7 of CTD recruits different transcription factors involved in transcription. In the current study, fluorescence anisotropy, pull down assay and molecular dynamics simulation studies employed to conclude that peptidyl-prolyl cis/trans-isomerase Rrd1 has strong affinity for unphosphorylated CTD rather than phosphorylated CTD for mRNA transcription. Rrd1 preferentially interacts with unphosphorylated GST-CTD in comparison to hyperphosphorylated GST-CTD in vitro. Fluorescence anisotropy revealed that recombinant Rrd1 prefers to bind unphosphorylated CTD peptide in comparison to phosphorylated CTD peptide. In computational studies, the RMSD of Rrd1-unphosphorylated CTD complex was greater than the RMSD of Rrd1-pCTD complex. During 50 ns MD simulation run Rrd1-pCTD complex get dissociated twice viz. 20 ns to 30 ns and 40 ns to 50 ns, while Rrd1-unpCTD complex remain stable throughout the process. Additionally, the Rrd1-unphosphorylated CTD complexes acquire comparatively higher number of H-bonds, water bridges and hydrophobic interactions occupancy than Rrd1-pCTD complex, concludes that the Rrd1 interacts more strongly with the unphosphorylated CTD than the pCTD.

Mediator structure and function in transcription initiation.

Recent advances in cryo-electron microscopy have led to multiple structures of Mediator in complex with the RNA polymerase II (Pol II) transcription initiation machinery. As a result we now hold in hands near-complete structures of both yeast and human Mediator complexes and have a better understanding of their interactions with the Pol II pre-initiation complex (PIC). Herein, we provide a summary of recent achievements and discuss their implications for future studies of Mediator and its role in gene regulation.

Effects of Histone H2B Ubiquitylations and H3K79me3 on Transcription Elongation.

Post-translational modifications of histone proteins often mediate gene regulation by altering the global and local stability of the nucleosome, the basic gene-packing unit of eukaryotes. We employed semisynthetic approaches to introduce histone H2B ubiquitylations at K34 (H2BK34ub) and K120 (H2BK120ub) and H3K79 trimethylation (H3K79me3). With these modified histones, we investigated their effects on the kinetics of transcription elongation by RNA polymerase II (Pol II) using single-molecule FRET. Pol II pauses at several locations within the nucleosome for a few seconds to minutes, which governs the overall transcription efficiency. We found that H2B ubiquitylations suppress pauses and shorten the pause durations near the nucleosome entry while H3K79me3 shortens the pause durations and increases the rate of RNA elongation near the center of the nucleosome. We also found that H2BK34ub facilitates partial rewrapping of the nucleosome upon Pol II passage. These observations suggest that H2B ubiquitylations promote transcription elongation and help maintain the chromatin structure by inducing and stabilizing nucleosome intermediates and that H3K79me3 facilitates Pol II progression possibly by destabilizing the local structure of the nucleosome. Our results provide the mechanisms of how these modifications coupled by a network of regulatory proteins facilitate transcription in two different regions of the nucleosome and help maintain the chromatin structure during active transcription.