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Abstract Eukaryotic gene regulation and pre-mRNA transcription depend on the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II. Due to its highly repetitive, intrinsically disordered sequence, the CTD enables clustering and phase separation of Pol II. The molecular interactions that drive CTD phase separation and Pol II clustering are unclear. Here, we show that multivalent interactions involving tyrosine impart temperature- and concentration-dependent self-coacervation of the CTD. NMR spectroscopy, molecular ensemble calculations and all-atom molecular dynamics simulations demonstrate the presence of diverse tyrosine-engaging interactions, including tyrosine-proline contacts, in condensed states of human CTD and other low-complexity proteins. We further show that the network of multivalent interactions involving tyrosine is responsible for the co-recruitment of the human Mediator complex and CTD during phase separation. Our work advances the understanding of the driving forces of CTD phase separation and thus provides the basis to better understand CTD-mediated Pol II clustering in eukaryotic gene transcription.

Abstract Mouse embryonic stem cells (mESCs) can adopt naïve, ground, and paused pluripotent states that give rise to unique transcriptomes. Here, we use transient transcriptome sequencing (TT‐seq) to define both coding and non‐coding transcription units (TUs) in these three pluripotent states and combine TT‐seq with RNA polymerase II occupancy profiling to unravel the kinetics of RNA metabolism genome‐wide. Compared to the naïve state (serum), RNA synthesis and turnover rates are globally reduced in the ground state (2i) and the paused state (mTORi). The global reduction in RNA synthesis goes along with a genome‐wide decrease of polymerase elongation velocity, which is related to epigenomic features and alterations in the Pol II termination window. Our data suggest that transcription activity is the main determinant of steady state mRNA levels in the naïve state and that genome‐wide changes in transcription kinetics invoke ground and paused pluripotent states.

Jochheim et al report an alternative, dimeric form of the coronavirus SARS-CoV-2 RNA dependent RNA polymerase (RdRp), which is used to replicate and transcribe its genome. They resolve its structure at 5.5 Å resolution and speculate that the dimer facilitates template switching during production of sub-genomic RNAs.

Although chromatin remodelers have been shown to align nucleosome arrays to barriers and to generate spacing regularity among nucleosomes within arrays, it has remained unclear how the distance to barrier and the spacing length are determined in absolute terms. Here, the authors reveal that remodelers contain a ‘ruler’ element that sets remodeler-specific alignment and spacing distances when generating nucleosome arrays.

RNA polymerase II needs to be imported into the nucleus to perform transcription. Fianu et al. show that RPAP2 binds to Pol II and enters the nucleus with it, before releasing it to perform transcription and returning to the cytoplasm.

Abstract The growth of human cancer cells is driven by aberrant enhancer and gene transcription activity. Here, we use transient transcriptome sequencing (TT‐seq) to map thousands of transcriptionally active putative enhancers in fourteen human cancer cell lines covering seven types of cancer. These enhancers were associated with cell type‐specific gene expression, enriched for genetic variants that predispose to cancer, and included functionally verified enhancers. Enhancer–promoter (E–P) pairing by correlation of transcription activity revealed ~ 40,000 putative E–P pairs, which were depleted for housekeeping genes and enriched for transcription factors, cancer‐associated genes, and 3D conformational proximity. The cell type specificity and transcription activity of target genes increased with the number of paired putative enhancers. Our results represent a rich resource for future studies of gene regulation by enhancers and their role in driving cancerous cell growth.

Remdesivir is a nucleoside analog that inhibits the SARS-CoV-2 RNA dependent RNA polymerase (RdRp) and is used as a drug to treat COVID19 patients. Here, the authors provide insights into the mechanism of remdesivir-induced RdRp stalling by determining the cryo-EM structures of SARS-CoV-2 RdRp with bound RNA molecules that contain remdesivir at defined positions and observe that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation.

The transcription factor Oct4 is essential for the maintenance and induction of stem cell pluripotency, but its functional roles are not fully understood. Here, we investigate the functions of Oct4 by depleting and subsequently recovering it in mouse embryonic stem cells (ESCs) and conducting a time-resolved multiomics analysis. Oct4 depletion leads to an immediate loss of its binding to enhancers, accompanied by a decrease in mRNA synthesis from its target genes that are part of the transcriptional network that maintains pluripotency. Gradual decrease of Oct4 binding to enhancers does not immediately change the chromatin accessibility but reduces transcription of enhancers. Conversely, partial recovery of Oct4 expression results in a rapid increase in chromatin accessibility, whereas enhancer transcription does not fully recover. These results indicate different concentration-dependent activities of Oct4. Whereas normal ESC levels of Oct4 are required for transcription of pluripotency enhancers, low levels of Oct4 are sufficient to retain chromatin accessibility, likely together with other factors such as Sox2.

AbstractAlu elements are one of the most successful groups of RNA retrotransposons and make up 11% of the human genome with over 1 million individual loci. They are linked to genetic defects, increases in sequence diversity, and influence transcriptional activity. Still, their RNA metabolism is poorly understood yet. It is even unclear whether Alu elements are mostly transcribed by RNA Polymerase II or III. We have conducted a transcription shutoff experiment by α-amanitin and metabolic RNA labeling by 4-thiouridine combined with RNA fragmentation (TT-seq) and RNA-seq to shed further light on the origin and life cycle of Alu transcripts. We find that Alu RNAs are more stable than previously thought and seem to originate in part from RNA Polymerase II activity, as previous reports suggest. Their expression however seems to be independent of the transcriptional activity of adjacent genes. Furthermore, we have developed a novel statistical test for detecting the expression of quantitative trait loci in Alu elements that relies on the de Bruijn graph representation of all Alu sequences. It controls for both statistical significance and biological relevance using a tuned k

Gene activation requires an increase of successful initiation events. Here, by employing a genome-wide kinetic analysis of transcription, the authors showed that gene activation generally requires a decrease in RNA Polymerase II (Pol II) promoter-proximal pausing while transcription of enhancer elements is not limited by Pol II pausing.

Sequencing of newly synthesised RNA can reveal the transcriptional dynamics in a population of cells. Here the authors develop NASC-seq to bring this sensitivity and temporal resolution to single-cell analysis.

The NER machinery contains the multisubunit transcription factor IIH (TFIIH) that opens the DNA repair bubble, scans for the lesion, and coordinates excision of the damaged site. Here the authors resolve the cryo-electron microscopy structure of the human core TFIIH-XPA-DNA complex and provide insights into its activation.

Eukaryotic transcription requires passage of RNA polymerase II (Pol II) through chromatin, which is impaired by nucleosomes. Here the authors report the cryo-EM structure of transcribing Pol II engaged with a downstream nucleosome core particle at an overall resolution of 4.4 Å, providing insights into the mechanism of chromatin transcription.

Enhancer activity drives cell differentiation and cell fate determination, but it remains unclear how enhancers cooperate during these processes. Here we investigate enhancer cooperation during transdifferentiation of human leukemia B-cells to macrophages. Putative enhancers are established by binding of the pioneer factor C/EBPα followed by chromatin opening and enhancer RNA (eRNA) synthesis from H3K4-monomethylated regions. Using eRNA synthesis as a proxy for enhancer activity, we find that most putative enhancers cooperate in an additive way to regulate transcription of assigned target genes. However, transcription from 136 target genes depends exponentially on the summed activity of its putative paired enhancers, indicating that these enhancers cooperate synergistically. The target genes are cell type-specific, suggesting that enhancer synergy can contribute to cell fate determination. Enhancer synergy appears to depend on cell type-specific transcription factors, and such interacting enhancers are not predicted from occupancy or accessibility data that are used to detect superenhancers.

Chromatin remodeling plays important roles in gene regulation during development, differentiation and in disease. The chromatin remodeling enzyme CHD4 is a component of the NuRD and ChAHP complexes that are involved in gene repression. Here, we report the cryo-electron microscopy (cryo-EM) structure of Homo sapiens CHD4 engaged with a nucleosome core particle in the presence of the non-hydrolysable ATP analogue AMP-PNP at an overall resolution of 3.1 Å. The ATPase motor of CHD4 binds and distorts nucleosomal DNA at superhelical location (SHL) +2, supporting the ‘twist defect’ model of chromatin remodeling. CHD4 does not induce unwrapping of terminal DNA, in contrast to its homologue Chd1, which functions in gene activation. Our structure also maps CHD4 mutations that are associated with human cancer or the intellectual disability disorder Sifrim-Hitz-Weiss syndrome.

The Paf1 complex (Paf1C) is an elongation factor assembly that forms the interface between transcribing Pol II and chromatin factors. Here the authors describe the architecture of Paf1C and its interface with Pol II, and show that Paf1C is globally required for normal mRNA transcription in yeast.

Termination of RNA polymerase II is a critical step in transcription. Here, the authors provide evidence that the fission yeast CID-RRM protein Seb1 is required for termination of coding and non-coding genes through interaction with both the polymerase and the nascent RNA.

Abstract To monitor transcriptional regulation in human cells, rapid changes in enhancer and promoter activity must be captured with high sensitivity and temporal resolution. Here, we show that the recently established protocol TT‐seq (“transient transcriptome sequencing”) can monitor rapid changes in transcription from enhancers and promoters during the immediate response of T cells to ionomycin and phorbol 12‐myristate 13‐acetate (PMA). TT‐seq maps eRNAs and mRNAs every 5 min after T‐cell stimulation with high sensitivity and identifies many new primary response genes. TT‐seq reveals that the synthesis of 1,601 eRNAs and 650 mRNAs changes significantly within only 15 min after stimulation, when standard RNA‐seq does not detect differentially expressed genes. Transcription of enhancers that are primed for activation by nucleosome depletion can occur immediately and simultaneously with transcription of target gene promoters. Our results indicate that enhancer transcription is a good proxy for enhancer regulatory activity in target gene activation, and establish TT‐seq as a tool for monitoring the dynamics of enhancer landscapes and transcription programs during cellular responses and differentiation.

Background: Specific regulation of gene expression determines cellular life and fate. Hundreds of transcription factor proteins (TFs) play critical role in this process. Typically, TFs bind short DNA motifs and then alter expression of their target genes. Majority of cellular DNA is associated with histones, as nucleosomes and higher order arrays, which occlude binding of most TFs. Only a minority of TFs (the pioneering TFs) are able to bind compact chromatin, and thus play a major role in altering gene expression and changing developmental programs. For instance, Oct4, Sox2 Klf4, c-Myc are highly expressed in embryonic stem cells and are able to drive differentiated somatic cells into pluripotent state. A recent study systematically explored interactions between the nucleosome and 220 TFs, and showed that most TFs preferably bind free DNA, and very few are able to bind nucleosomes in vitro. This study has also provided a selection of DNA-templates selected for ability to form nucleosomes and promote binding of a TF of interest. Results and methods: Currently our mechanistic understanding of how pioneer TFs interact with chromatin is rather poor. Our structural knowledge in that area is limited to individual TFs or TFs bound to short DNA fragments. Thus, we aimed at structural characterization of pioneer TFs in the context of a nucleosome. We have formed a complex of a nucleosome on a DNA template from with a pioneer TF from purified individual components and have solved the structure by cryo-EM. We have confirmed the binding of a pioneer TF to the nucleosome by electrophoretic mobility shift assays. Conclusions: We report the first structure of a nucleosome and a pioneer TF. Our structural and biochemical results illustrate a possible role of a pioneer TF in nucleosome destabilization and shed light on how such TFs can interact with chromatin.

RNA degradation pathways enable RNA processing, the regulation of RNA levels, and the surveillance of aberrant or poorly functional RNAs in cells. Here we provide transcriptome-wide RNA-binding profiles of 30 general RNA degradation factors in the yeast Saccharomyces cerevisiae. The profiles reveal the distribution of degradation factors between different RNA classes. They are consistent with the canonical degradation pathway for closed-loop forming mRNAs after deadenylation. Modeling based on mRNA half-lives suggests that most degradation factors bind intact mRNAs, whereas decapping factors are recruited only for mRNA degradation, consistent with decapping being a rate-limiting step. Decapping factors preferentially bind mRNAs with non-optimal codons, consistent with rapid degradation of inefficiently translated mRNAs. Global analysis suggests that the nuclear surveillance machinery, including the complexes Nrd1/Nab3 and TRAMP4, targets aberrant nuclear RNAs and processes snoRNAs.

RNA splicing is an essential part of eukaryotic gene expression. Although the mechanism of splicing has been extensively studied in vitro, in vivo kinetics for the two-step splicing reaction remain poorly understood. Here, we combine transient transcriptome sequencing (TT-seq) and mathematical modeling to quantify RNA metabolic rates at donor and acceptor splice sites across the human genome. Splicing occurs in the range of minutes and is limited by the speed of RNA polymerase elongation. Splicing kinetics strongly depends on the position and nature of nucleotides flanking splice sites, and on structural interactions between unspliced RNA and small nuclear RNAs in spliceosomal intermediates. Finally, we introduce the ‘yield’ of splicing as the efficiency of converting unspliced to spliced RNA and show that it is highest for mRNAs and independent of splicing kinetics. These results lead to quantitative models describing how splicing rates and yield are encoded in the human genome.

RNA polymerase I is the central enzyme that synthesizes ribosomal RNA in eukaryotic cells, and its regulation underlies cell growth. Here the authors present a high-resolution cryo-EM structure of the Pol I-Rrn3 complex that explains how Rrn3 specifically recognizes Pol I to form an initiation competent complex.

Abstract To decrypt the regulatory code of the genome, sequence elements must be defined that determine the kinetics of RNA metabolism and thus gene expression. Here, we attempt such decryption in an eukaryotic model organism, the fission yeast S. pombe. We first derive an improved genome annotation that redefines borders of 36% of expressed mRNAs and adds 487 non‐coding RNAs (ncRNAs). We then combine RNA labeling in vivo with mathematical modeling to obtain rates of RNA synthesis and degradation for 5,484 expressed RNAs and splicing rates for 4,958 introns. We identify functional sequence elements in DNA and RNA that control RNA metabolic rates and quantify the contributions of individual nucleotides to RNA synthesis, splicing, and degradation. Our approach reveals distinct kinetics of mRNA and ncRNA metabolism, separates antisense regulation by transcription interference from RNA interference, and provides a general tool for studying the regulatory code of genomes.

Abstract DNA replication, transcription and repair involve the recruitment of protein complexes that change their composition as they progress along the genome in a directed or strand‐specific manner. Chromatin immunoprecipitation in conjunction with hidden Markov models (HMMs) has been instrumental in understanding these processes, as they segment the genome into discrete states that can be related to DNA‐associated protein complexes. However, current HMM‐based approaches are not able to assign forward or reverse direction to states or properly integrate strand‐specific (e.g., RNA expression) with non‐strand‐specific (e.g., ChIP) data, which is indispensable to accurately characterize directed processes. To overcome these limitations, we introduce bidirectional HMMs which infer directed genomic states from occupancy profiles de novo. Application to RNA polymerase II‐associated factors in yeast and chromatin modifications in human T cells recovers the majority of transcribed loci, reveals gene‐specific variations in the yeast transcription cycle and indicates the existence of directed chromatin state patterns at transcribed, but not at repressed, regions in the human genome. In yeast, we identify 32 new transcribed loci, a regulated initiation–elongation transition, the absence of elongation factors Ctk1 and Paf1 from a class of genes, a distinct transcription mechanism for highly expressed genes and novel DNA sequence motifs associated with transcription termination. We anticipate bidirectional HMMs to significantly improve the analyses of genome‐associated directed processes.

Gene transcription can be activated by decreasing the duration of RNA polymerase II pausing in the promoter-proximal region, but how this is achieved remains unclear. Here we use a ‘multi-omics’ approach to demonstrate that the duration of polymerase pausing generally limits the productive frequency of transcription initiation in human cells (‘pause-initiation limit’). We further engineer a human cell line to allow for specific and rapid inhibition of the P-TEFb kinase CDK9, which is implicated in polymerase pause release. CDK9 activity decreases the pause duration but also increases the productive initiation frequency. This shows that CDK9 stimulates release of paused polymerase and activates transcription by increasing the number of transcribing polymerases and thus the amount of mRNA synthesized per time. CDK9 activity is also associated with long-range chromatin interactions, suggesting that enhancers can influence the pause-initiation limit to regulate transcription.

For transcription through chromatin, RNA polymerase (Pol) II associates with elongation factors (EFs). Here we show that many EFs crosslink to RNA emerging from transcribing Pol II in the yeast Saccharomyces cerevisiae. Most EFs crosslink preferentially to mRNAs, rather than unstable non-coding RNAs. RNA contributes to chromatin association of many EFs, including the Pol II serine 2 kinases Ctk1 and Bur1 and the histone H3 methyltransferases Set1 and Set2. The Ctk1 kinase complex binds RNA in vitro, consistent with direct EF-RNA interaction. Set1 recruitment to genes in vivo depends on its RNA recognition motifs (RRMs). These results strongly suggest that nascent RNA contributes to EF recruitment to transcribing Pol II. We propose that EF-RNA interactions facilitate assembly of the elongation complex on transcribed genes when RNA emerges from Pol II, and that loss of EF-RNA interactions upon RNA cleavage at the polyadenylation site triggers disassembly of the elongation complex.

Accurate maps of promoters and enhancers are required for understanding transcriptional regulation. Promoters and enhancers are usually mapped by integration of chromatin assays charting histone modifications, DNA accessibility, and transcription factor binding. However, current algorithms are limited by unrealistic data distribution assumptions. Here we propose GenoSTAN (Genomic STate ANnotation), a hidden Markov model overcoming these limitations. We map promoters and enhancers for 127 cell types and tissues from the ENCODE and Roadmap Epigenomics projects, today's largest compendium of chromatin assays. Extensive benchmarks demonstrate that GenoSTAN generally identifies promoters and enhancers with significantly higher accuracy than previous methods. Moreover, GenoSTAN-derived promoters and enhancers showed significantly higher enrichment of complex trait-associated genetic variants than current annotations. Altogether, GenoSTAN provides an easy-to-use tool to define promoters and enhancers in any system, and our annotation of human transcriptional cis-regulatory elements constitutes a rich resource for future research in biology and medicine.

Abstract During the cell cycle, the levels of hundreds of mRNAs change in a periodic manner, but how this is achieved by alterations in the rates of mRNA synthesis and degradation has not been studied systematically. Here, we used metabolic RNA labeling and comparative dynamic transcriptome analysis (cDTA) to derive mRNA synthesis and degradation rates every 5 min during three cell cycle periods of the yeast Saccharomyces cerevisiae. A novel statistical model identified 479 genes that show periodic changes in mRNA synthesis and generally also periodic changes in their mRNA degradation rates. Peaks of mRNA degradation generally follow peaks of mRNA synthesis, resulting in sharp and high peaks of mRNA levels at defined times during the cell cycle. Whereas the timing of mRNA synthesis is set by upstream DNA motifs and their associated transcription factors (TFs), the synthesis rate of a periodically expressed gene is apparently set by its core promoter.

To quantify gene regulation, a function is required that relates transcription factor binding to DNA (input) to the rate of mRNA synthesis from a target gene (output). Such a ‘gene regulation function’ (GRF) generally cannot be measured because the experimental titration of inputs and simultaneous readout of outputs is difficult. Here we show that GRFs may instead be inferred from natural changes in cellular gene expression, as exemplified for the cell cycle in the yeast S. cerevisiae. We develop this inference approach based on a time series of mRNA synthesis rates from a synchronized population of cells observed over three cell cycles. We first estimate the functional form of how input transcription factors determine mRNA output and then derive GRFs for target genes in the CLB2 gene cluster that are expressed during G2/M phase. Systematic analysis of additional GRFs suggests a network architecture that rationalizes transcriptional cell cycle oscillations. We find that a transcription factor network alone can produce oscillations in mRNA expression, but that additional input from cyclin oscillations is required to arrive at the native behaviour of the cell cycle oscillator.

Transcription regulation in metazoans often involves promoter-proximal pausing of RNA polymerase (Pol) II, which requires the 4-subunit negative elongation factor (NELF). Here we discern the functional architecture of human NELF through X-ray crystallography, protein crosslinking, biochemical assays, and RNA crosslinking in cells. We identify a NELF core subcomplex formed by conserved regions in subunits NELF-A and NELF-C, and resolve its crystal structure. The NELF-AC subcomplex binds single-stranded nucleic acids in vitro, and NELF-C associates with RNA in vivo. A positively charged face of NELF-AC is involved in RNA binding, whereas the opposite face of the NELF-AC subcomplex binds NELF-B. NELF-B is predicted to form a HEAT repeat fold, also binds RNA in vivo, and anchors the subunit NELF-E, which is confirmed to bind RNA in vivo. These results reveal the three-dimensional architecture and three RNA-binding faces of NELF.

Abstract To obtain rates of mRNA synthesis and decay in yeast, we established dynamic transcriptome analysis (DTA). DTA combines non‐perturbing metabolic RNA labeling with dynamic kinetic modeling. DTA reveals that most mRNA synthesis rates are around several transcripts per cell and cell cycle, and most mRNA half‐lives range around a median of 11 min. DTA can monitor the cellular response to osmotic stress with higher sensitivity and temporal resolution than standard transcriptomics. In contrast to monotonically increasing total mRNA levels, DTA reveals three phases of the stress response. During the initial shock phase, mRNA synthesis and decay rates decrease globally, resulting in mRNA storage. During the subsequent induction phase, both rates increase for a subset of genes, resulting in production and rapid removal of stress‐responsive mRNAs. During the recovery phase, decay rates are largely restored, whereas synthesis rates remain altered, apparently enabling growth at high salt concentration. Stress‐induced changes in mRNA synthesis rates are predicted from gene occupancy with RNA polymerase II. DTA‐derived mRNA synthesis rates identified 16 stress‐specific pairs/triples of cooperative transcription factors, of which seven were known. Thus, DTA realistically monitors the dynamics in mRNA metabolism that underlie gene regulatory systems.

Monoclonal antibodies (mAbs) and proteins containing antibody domains are the most prevalent class of biotherapeutics in diverse indication areas. Today, established techniques such as immunization or phage display allow for an efficient generation of new mAbs. Besides functional properties, the stability of future therapeutic mAbs is a key selection criterion which is essential for the development of a drug candidate into a marketed product. Therapeutic proteins may degrade via asparagine (Asn) deamidation and aspartate (Asp) isomerization, but the factors responsible for such degradation remain poorly understood. We studied the structural properties of a large, uniform dataset of Asn and Asp residues in the variable domains of antibodies. Their structural parameters were correlated with the degradation propensities measured by mass spectrometry. We show that degradation hotspots can be characterized by their conformational flexibility, the size of the C-terminally flanking amino acid residue, and secondary structural parameters. From these results we derive an accurate in silico prediction method for the degradation propensity of both Asn and Asp residues in the complementarity-determining regions (CDRs) of mAbs.

Messenger RNA (mRNA) synthesis and export are tightly linked, but the molecular mechanisms of this coupling are largely unknown. In Saccharomyces cerevisiae, the conserved TREX complex couples transcription to mRNA export and mediates mRNP formation. Here, we show that TREX is recruited to the transcription machinery by direct interaction of its subcomplex THO with the serine 2-serine 5 (S2/S5) diphosphorylated CTD of RNA polymerase II. S2 and/or tyrosine 1 (Y1) phosphorylation of the CTD is required for TREX occupancy in vivo, establishing a second interaction platform necessary for TREX recruitment in addition to RNA. Genome-wide analyses show that the occupancy of THO and the TREX components Sub2 and Yra1 increases from the 5' to the 3' end of the gene in accordance with the CTD S2 phosphorylation pattern. Importantly, in a mutant strain, in which TREX is recruited to genes but does not increase towards the 3' end, the expression of long transcripts is specifically impaired. Thus, we show for the first time that a 5'-3' increase of a protein complex is essential for correct expression of the genome. In summary, we provide insight into how the phospho-code of the CTD directs mRNP formation and export through TREX recruitment.

The Mediator is a highly conserved, large multiprotein complex that is involved essentially in the regulation of eukaryotic mRNA transcription. It acts as a general transcription factor by integrating regulatory signals from gene-specific activators or repressors to the RNA Polymerase II. The internal network of interactions between Mediator subunits that conveys these signals is largely unknown. Here, we introduce MC EMiNEM, a novel method for the retrieval of functional dependencies between proteins that have pleiotropic effects on mRNA transcription. MC EMiNEM is based on Nested Effects Models (NEMs), a class of probabilistic graphical models that extends the idea of hierarchical clustering. It combines mode-hopping Monte Carlo (MC) sampling with an Expectation-Maximization (EM) algorithm for NEMs to increase sensitivity compared to existing methods. A meta-analysis of four Mediator perturbation studies in Saccharomyces cerevisiae, three of which are unpublished, provides new insight into the Mediator signaling network. In addition to the known modular organization of the Mediator subunits, MC EMiNEM reveals a hierarchical ordering of its internal information flow, which is putatively transmitted through structural changes within the complex. We identify the N-terminus of Med7 as a peripheral entity, entailing only local structural changes upon perturbation, while the C-terminus of Med7 and Med19 appear to play a central role. MC EMiNEM associates Mediator subunits to most directly affected genes, which, in conjunction with gene set enrichment analysis, allows us to construct an interaction map of Mediator subunits and transcription factors.

In eukaryotes, hundreds of mRNAs are localized by specialized transport complexes. For localization, transcripts are recognized by RNA-binding proteins and incorporated into motor-containing messenger ribonucleoprotein particles (mRNPs). To date, the molecular assembly of such mRNPs is not well understood and most details on cargo specificity remain unresolved. We used ASH1-mRNA transport in yeast to provide a first assessment of where and how localizing mRNAs are specifically recognized and incorporated into mRNPs. By using in vitro-interaction and reconstitution assays, we found that none of the implicated mRNA-binding proteins showed highly specific cargo binding. Instead, we identified the cytoplasmic myosin adapter She3p as additional RNA-binding protein. We further found that only the complex of the RNA-binding proteins She2p and She3p achieves synergistic cargo binding, with an at least 60-fold higher affinity for localizing mRNAs when compared to control RNA. Mutational studies identified a C-terminal RNA-binding fragment of She3p to be important for synergistic RNA binding with She2p. The observed cargo specificity of the ternary complex is considerably higher than previously reported for localizing mRNAs. It suggests that RNA binding for mRNP localization generally exhibits higher selectivity than inferred from previous in vitro data. This conclusion is fully consistent with a large body of in vivo evidence from different organisms. Since the ternary yeast complex only assembles in the cytoplasm, specific mRNA recognition might be limited to the very last steps of mRNP assembly. Remarkably, the mRNA itself triggers the assembly of mature, motor-containing complexes. Our reconstitution of a major portion of the mRNA-transport complex offers new and unexpected insights into the molecular assembly of specific, localization-competent mRNPs and provides an important step forward in our mechanistic understanding of mRNA localization in general.

Initiation of RNA polymerase (Pol) II transcription requires assembly of the pre-initiation complex (PIC) at the promoter. In the classical view, PIC assembly starts with binding of the TATA box-binding protein (TBP) to the TATA box. However, a TATA box occurs in only 15% of promoters in the yeast Saccharomyces cerevisiae, posing the question how most yeast promoters nucleate PIC assembly. Here we show that one third of all yeast promoters contain a novel conserved DNA element, the GA element (GAE), that generally does not co-occur with the TATA box. The distance of the GAE to the transcription start site (TSS) resembles the distance of the TATA box to the TSS. The TATA-less TMT1 core promoter contains a GAE, recruits TBP, and supports formation of a TBP-TFIIB-DNA-complex. Mutation of the promoter region surrounding the GAE abolishes transcription in vivo and in vitro. A 32-nucleotide promoter region containing the GAE can functionally substitute for the TATA box in a TATA-containing promoter. This identifies the GAE as a conserved promoter element in TATA-less promoters.