Rho is a general transcription termination factor playing essential roles in RNA polymerase (RNAP) recycling, gene regulation, and genomic stability in most bacteria. Traditional models of ...transcription termination postulate that hexameric Rho loads onto RNA prior to contacting RNAP and then translocates along the transcript in pursuit of the moving RNAP to pull RNA from it. Here, we report the cryoelectron microscopy (cryo-EM) structures of two termination process intermediates. Prior to interacting with RNA, Rho forms a specific “pre-termination complex” (PTC) with RNAP and elongation factors NusA and NusG, which stabilize the PTC. RNA exiting RNAP interacts with NusA before entering the central channel of Rho from the distal C-terminal side of the ring. We map the principal interactions in the PTC and demonstrate their critical role in termination. Our results support a mechanism in which the formation of a persistent PTC is a prerequisite for termination.
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•ρ binds to RNAP first, not to the RNA, thus forming the pre-termination complex (PTC)•PTC structure reveals specific RNAP-ρ interactions that are important for termination•NusG interacts with open-ring ρ in PTC, thus promoting ρ-dependent termination•NusA stabilizes PTC and directs the nascent RNA into the central channel of ρ ring
Hao et al. report the structural view of the transcription termination complex on its path to termination. Combining the high-resolution structural information with new biochemical, protein chemical, and genetic data, it offers a comprehensive molecular mechanism of factor-dependent transcription termination, which dramatically deviates from the textbook paradigm.
Eukaryotic translation factors, especially initiation factors have garnered much attention with regards to their role in the onset and progression of different cancers. However, the expression levels ...and prognostic significance of translation elongation factors remain poorly explored in different cancers. In this study, we have investigated the mRNA transcript levels of seven translation elongation factors in different cancer types using Oncomine and TCGA databases. Furthermore, we have identified the prognostic significance of these factors using Kaplan-Meier Plotter and SurvExpress databases. We observed altered expression levels of all the elongation factors in different cancers. Higher expression of EEF1A2, EEF1B2, EEF1G, EEF1D, EEF1E1 and EEF2 was observed in most of the cancer types, whereas reverse trend was observed for EEF1A1. Overexpression of many factors predicted poor prognosis in breast (EEF1D, EEF1E1, EEF2) and lung cancer (EEF1A2, EEF1B2, EEF1G, EEF1E1). However, we didn't see any common correlation of expression levels of elongation factors with survival outcomes across cancer types. Cancer subtype stratification showed association of survival outcomes and expression levels of elongation factors in specific sub-types of breast, lung and gastric cancer. Most interestingly, we observed a reciprocal relationship between the expression levels of the two EEF1A isoforms viz. EEF1A1 and EEF1A2, in most of the cancer types. Our results suggest that translation elongation factors can have a role in tumorigenesis and affect survival in cancer specific manner. Elongation factors have potential to serve as biomarkers and therapeutic drug targets, yet further study is required. Reciprocal relationship of differential expression between EEF1A isoforms observed in multiple cancer types indicates opposing roles in cancer and needs further investigation.
Viia Kõiv, Liis Andresen, Martin Broberg, Jekaterina Frolova, Panu Somervuo, Petri Auvinen, E. Tapio Palva, Minna Pirhonen, Tanel Tenson, Andres Mäe Dr. Palva provided the DNA sequence information of ...Pectobacterium wasabiae SCC3193, and contributed to the microarray analyses for the study and to the analysis of the results. (2013) Correction: Lack of RsmA-Mediated Control Results in Constant Hypervirulence, Cell Elongation, and Hyperflagellation in Pectobacterium wasabiae.
The super elongation complex (SEC) containing positive transcription elongation factor b plays a critical role in regulating transcription elongation. AFF1 and AFF4, two members of the AF4/FMR2 ...family, act as central scaffold proteins of SEC and are associated with various human diseases. However, their precise roles in transcriptional control remain unclear. Here, we investigate differences in the genomic distribution patterns of AFF1 and AFF4 around transcription start sites (TSSs). AFF1 mainly binds upstream of the TSS, while AFF4 is enriched downstream of the TSS. Notably, disruption of AFF4 results in slow elongation and early termination in a subset of AFF4-bound active genes, whereas AFF1 deletion leads to fast elongation and transcriptional readthrough in the same subset of genes. Additionally, AFF1 knockdown increases AFF4 levels at chromatin, and vice versa. In summary, these findings demonstrate that AFF1 and AFF4 function antagonistically to regulate RNA polymerase II transcription.
Structural biology studies performed inside cells can capture molecular machines in action within their native context. In this work, we developed an integrative in-cell structural approach using the ...genome-reduced human pathogen
We combined whole-cell cross-linking mass spectrometry, cellular cryo-electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at subnanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpointed NusA at the interface between a NusG-bound elongating RNAP and the ribosome and propose that it can mediate transcription-translation coupling. Translation inhibition dissociated the expressome, whereas transcription inhibition stalled and rearranged it. Thus, the active expressome architecture requires both translation and transcription elongation within the cell.
Targeting translational factor proteins (TFPs) presents significant promise for the development of innovative antitubercular drugs. Previous insights from antibiotic binding mechanisms and recently ...solved 3D crystal structures of Mycobacterium tuberculosis (Mtb) elongation factor thermo unstable–GDP (EF-Tu–GDP), elongation factor thermo stable–EF-Tu (EF-Ts–EF-Tu), and elongation factor G–GDP (EF-G–GDP) have opened up new avenues for the design and development of potent antituberculosis (anti-TB) therapies.
Targeting translational factor proteins (TFPs) presents significant promise for the development of innovative antitubercular drugs. Previous insights from antibiotic binding mechanisms and recently solved 3D crystal structures of Mycobacterium tuberculosis (Mtb) elongation factor thermo unstable–GDP (EF-Tu–GDP), elongation factor thermo stable–EF-Tu (EF-Ts–EF-Tu), and elongation factor G–GDP (EF-G–GDP) have opened up new avenues for the design and development of potent antituberculosis (anti-TB) therapies.
The super elongation complex (SEC) is required for robust and productive transcription through release of RNA polymerase II (Pol II) with its P-TEFb module and promoting transcriptional processivity ...with its ELL2 subunit. Malfunction of SEC contributes to multiple human diseases including cancer. Here, we identify peptidomimetic lead compounds, KL-1 and its structural homolog KL-2, which disrupt the interaction between the SEC scaffolding protein AFF4 and P-TEFb, resulting in impaired release of Pol II from promoter-proximal pause sites and a reduced average rate of processive transcription elongation. SEC is required for induction of heat-shock genes and treating cells with KL-1 and KL-2 attenuates the heat-shock response from Drosophila to human. SEC inhibition downregulates MYC and MYC-dependent transcriptional programs in mammalian cells and delays tumor progression in a mouse xenograft model of MYC-driven cancer, indicating that small-molecule disruptors of SEC could be used for targeted therapy of MYC-induced cancer.
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•Discovery of small-molecule inhibitors of SEC and transcription elongation by Pol II•KL-1 and KL-2 disrupt the cyclin T1-AFF4 interaction within SEC•SEC inhibitors attenuate SEC-dependent rapid transcriptional responses•MYC transcriptional programs are inhibited by SEC chemical disruptors KL-1/KL-2
Targeting transcriptional elongation with small-molecule inhibitors of the super elongation complex blocks transcriptional programs driven by the oncogene MYC
In response to stress, human cells coordinately downregulate transcription and translation of housekeeping genes. To downregulate transcription, the negative elongation factor (NELF) is recruited to ...gene promoters impairing RNA polymerase II elongation. Here we report that NELF rapidly forms nuclear condensates upon stress in human cells. Condensate formation requires NELF dephosphorylation and SUMOylation induced by stress. The intrinsically disordered region (IDR) in NELFA is necessary for nuclear NELF condensation and can be functionally replaced by the IDR of FUS or EWSR1 protein. We find that biomolecular condensation facilitates enhanced recruitment of NELF to promoters upon stress to drive transcriptional downregulation. Importantly, NELF condensation is required for cellular viability under stressful conditions. We propose that stress-induced NELF condensates reported here are nuclear counterparts of cytosolic stress granules. These two stress-inducible condensates may drive the coordinated downregulation of transcription and translation, likely forming a critical node of the stress survival strategy.
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•Transcription regulator NELF undergoes stress-induced condensation in human cells•NELF condensation is regulated by its phosphorylation and SUMOylation•Intrinsically disordered region of NELFA is necessary to drive condensation•NELF condensation downregulates transcription to promote cell survival upon stress
Rawat et al. discovered stress-induced condensation of the transcriptional regulator NELF in human cells, recapitulated by phase separation of recombinant NELF in vitro. NELF condensation is governed by its disordered tentacles and stress-contingent changes in NELF phosphorylation and SUMOylation. NELF condensation facilitates stress-mediated transcriptional downregulation, supporting cell survival upon stress.
Recent advances in sequencing techniques that measure nascent transcripts and that reveal the positioning of RNA polymerase II (Pol II) have shown that the pausing of Pol II in promoter-proximal ...regions and its release to initiate a phase of productive elongation are key steps in transcription regulation. Moreover, after the release of Pol II from the promoter-proximal region, elongation rates are highly dynamic throughout the transcription of a gene, and vary on a gene-by-gene basis. Interestingly, Pol II elongation rates affect co-transcriptional processes such as splicing, termination and genome stability. Increasing numbers of factors and regulatory mechanisms have been associated with the steps of transcription elongation by Pol II, revealing that elongation is a highly complex process. Elongation is thus now recognized as a key phase in the regulation of transcription by Pol II.
The journey of RNA polymerase II (Pol II) as it transcribes a gene is anything but a smooth ride. Transcript elongation is discontinuous and can be perturbed by intrinsic regulatory barriers, such as ...promoter-proximal pausing, nucleosomes, RNA secondary structures and the underlying DNA sequence. More substantial blocking of Pol II translocation can be caused by other physiological circumstances and extrinsic obstacles, including other transcribing polymerases, the replication machinery and several types of DNA damage, such as bulky lesions and DNA double-strand breaks. Although numerous different obstacles cause Pol II stalling or arrest, the cell somehow distinguishes between them and invokes different mechanisms to resolve each roadblock. Resolution of Pol II blocking can be as straightforward as temporary backtracking and transcription elongation factor S-II (TFIIS)-dependent RNA cleavage, or as drastic as premature transcription termination or degradation of polyubiquitylated Pol II and its associated nascent RNA. In this Review, we discuss the current knowledge of how these different Pol II stalling contexts are distinguished by the cell, how they overlap with each other, how they are resolved and how, when unresolved, they can cause genome instability.
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