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Persistence of backtracking by human RNA polymerase II

Yang, Kevin B; Rasouly, Aviram; Epshtein, Vitaly; Martinez, Criseyda; Nguyen, Thao; Shamovsky, Ilya; Nudler, Evgeny
RNA polymerase II (RNA Pol II) can backtrack during transcription elongation, exposing the 3' end of nascent RNA. Nascent RNA sequencing can approximate the location of backtracking events that are quickly resolved; however, the extent and genome-wide distribution of more persistent backtracking are unknown. Consequently, we developed a method to directly sequence the extruded, "backtracked" 3' RNA. Our data show that RNA Pol II slides backward more than 20 nt in human cells and can persist in this backtracked state. Persistent backtracking mainly occurs where RNA Pol II pauses near promoters and intron-exon junctions and is enriched in genes involved in translation, replication, and development, where gene expression is decreased if these events are unresolved. Histone genes are highly prone to persistent backtracking, and the resolution of such events is likely required for timely expression during cell division. These results demonstrate that persistent backtracking can potentially affect diverse gene expression programs.
PMID: 38340716
ISSN: 1097-4164
CID: 5635502

General transcription factor from Escherichia coli with a distinct mechanism of action

Vasilyev, Nikita; Liu, Mengjie M J; Epshtein, Vitaly; Shamovsky, Ilya; Nudler, Evgeny
Gene expression in Escherichia coli is controlled by well-established mechanisms that activate or repress transcription. Here, we identify CedA as an unconventional transcription factor specifically associated with the RNA polymerase (RNAP) σ70 holoenzyme. Structural and biochemical analysis of CedA bound to RNAP reveal that it bridges distant domains of β and σ70 subunits to stabilize an open-promoter complex. CedA does so without contacting DNA. We further show that cedA is strongly induced in response to amino acid starvation, oxidative stress and aminoglycosides. CedA provides a basal level of tolerance to these clinically relevant antibiotics, as well as to rifampicin and peroxide. Finally, we show that CedA modulates transcription of hundreds of bacterial genes, which explains its pleotropic effect on cell physiology and pathogenesis.
PMID: 38177674
ISSN: 1545-9985
CID: 5624352

Bacterial histones unveiled

Pani, Bibhusita; Nudler, Evgeny
PMID: 37857818
ISSN: 2058-5276
CID: 5610402

A noncanonical function of SKP1 regulates the switch between autophagy and unconventional secretion

Li, Jie; Krause, Gregory J; Gui, Qi; Kaushik, Susmita; Rona, Gergely; Zhang, Qingyue; Liang, Feng-Xia; Dhabaria, Avantika; Anerillas, Carlos; Martindale, Jennifer L; Vasilyev, Nikita; Askenazi, Manor; Ueberheide, Beatrix; Nudler, Evgeny; Gorospe, Myriam; Cuervo, Ana Maria; Pagano, Michele
Intracellular degradation of proteins and organelles by the autophagy-lysosome system is essential for cellular quality control and energy homeostasis. Besides degradation, endolysosomal organelles can fuse with the plasma membrane and contribute to unconventional secretion. Here, we identify a function for mammalian SKP1 in endolysosomes that is independent of its established role as an essential component of the family of SCF/CRL1 ubiquitin ligases. We found that, under nutrient-poor conditions, SKP1 is phosphorylated on Thr131, allowing its interaction with V1 subunits of the vacuolar ATPase (V-ATPase). This event, in turn, promotes V-ATPase assembly to acidify late endosomes and enhance endolysosomal degradation. Under nutrient-rich conditions, SUMOylation of phosphorylated SKP1 allows its binding to and dephosphorylation by the PPM1B phosphatase. Dephosphorylated SKP1 interacts with SEC22B to promote unconventional secretion of the content of less acidified hybrid endosomal/autophagic compartments. Collectively, our study implicates SKP1 phosphorylation as a switch between autophagy and unconventional secretion in a manner dependent on cellular nutrient status.
PMID: 37831778
ISSN: 2375-2548
CID: 5604232

High-resolution landscape of an antibiotic binding site

Yang, Kevin B; Cameranesi, Maria; Gowder, Manjunath; Martinez, Criseyda; Shamovsky, Yosef; Epshtein, Vitaliy; Hao, Zhitai; Nguyen, Thao; Nirenstein, Eric; Shamovsky, Ilya; Rasouly, Aviram; Nudler, Evgeny
Antibiotic binding sites are located in important domains of essential enzymes and have been extensively studied in the context of resistance mutations; however, their study is limited by positive selection. Using multiplex genome engineering1 to overcome this constraint, we generate and characterize a collection of 760 single-residue mutants encompassing the entire rifampicin binding site of Escherichia coli RNA polymerase (RNAP). By genetically mapping drug-enzyme interactions, we identify an alpha helix where mutations considerably enhance or disrupt rifampicin binding. We find mutations in this region that prolong antibiotic binding, converting rifampicin from a bacteriostatic to bactericidal drug by inducing lethal DNA breaks. The latter are replication dependent, indicating that rifampicin kills by causing detrimental transcription-replication conflicts at promoters. We also identify additional binding site mutations that greatly increase the speed of RNAP.Fast RNAP depletes the cell of nucleotides, alters cell sensitivity to different antibiotics and provides a cold growth advantage. Finally, by mapping natural rpoB sequence diversity, we discover that functional rifampicin binding site mutations that alter RNAP properties or confer drug resistance occur frequently in nature.
PMID: 37648864
ISSN: 1476-4687
CID: 5618342

Catalytic and non-catalytic mechanisms of histone H4 lysine 20 methyltransferase SUV420H1

Abini-Agbomson, Stephen; Gretarsson, Kristjan; Shih, Rochelle M; Hsieh, Laura; Lou, Tracy; De Ioannes, Pablo; Vasilyev, Nikita; Lee, Rachel; Wang, Miao; Simon, Matthew D; Armache, Jean-Paul; Nudler, Evgeny; Narlikar, Geeta; Liu, Shixin; Lu, Chao; Armache, Karim-Jean
SUV420H1 di- and tri-methylates histone H4 lysine 20 (H4K20me2/H4K20me3) and plays crucial roles in DNA replication, repair, and heterochromatin formation. It is dysregulated in several cancers. Many of these processes were linked to its catalytic activity. However, deletion and inhibition of SUV420H1 have shown distinct phenotypes, suggesting that the enzyme likely has uncharacterized non-catalytic activities. Our cryoelectron microscopy (cryo-EM), biochemical, biophysical, and cellular analyses reveal how SUV420H1 recognizes its nucleosome substrates, and how histone variant H2A.Z stimulates its catalytic activity. SUV420H1 binding to nucleosomes causes a dramatic detachment of nucleosomal DNA from the histone octamer, which is a non-catalytic activity. We hypothesize that this regulates the accessibility of large macromolecular complexes to chromatin. We show that SUV420H1 can promote chromatin condensation, another non-catalytic activity that we speculate is needed for its heterochromatin functions. Together, our studies uncover and characterize the catalytic and non-catalytic mechanisms of SUV420H1, a key histone methyltransferase that plays an essential role in genomic stability.
PMID: 37595555
ISSN: 1097-4164
CID: 5598082

Structural basis of histone H2A lysine 119 deubiquitination by Polycomb repressive deubiquitinase BAP1/ASXL1

Thomas, Jonathan F; Valencia-Sánchez, Marco Igor; Tamburri, Simone; Gloor, Susan L; Rustichelli, Samantha; Godínez-López, Victoria; De Ioannes, Pablo; Lee, Rachel; Abini-Agbomson, Stephen; Gretarsson, Kristjan; Burg, Jonathan M; Hickman, Allison R; Sun, Lu; Gopinath, Saarang; Taylor, Hailey F; Sun, Zu-Wen; Ezell, Ryan J; Vaidya, Anup; Meiners, Matthew J; Cheek, Marcus A; Rice, William J; Svetlov, Vladimir; Nudler, Evgeny; Lu, Chao; Keogh, Michael-Christopher; Pasini, Diego; Armache, Karim-Jean
Histone H2A lysine 119 (H2AK119Ub) is monoubiquitinated by Polycomb repressive complex 1 and deubiquitinated by Polycomb repressive deubiquitinase complex (PR-DUB). PR-DUB cleaves H2AK119Ub to restrict focal H2AK119Ub at Polycomb target sites and to protect active genes from aberrant silencing. The PR-DUB subunits (BAP1 and ASXL1) are among the most frequently mutated epigenetic factors in human cancers. How PR-DUB establishes specificity for H2AK119Ub over other nucleosomal ubiquitination sites and how disease-associated mutations of the enzyme affect activity are unclear. Here, we determine a cryo-EM structure of human BAP1 and the ASXL1 DEUBAD in complex with a H2AK119Ub nucleosome. Our structural, biochemical, and cellular data reveal the molecular interactions of BAP1 and ASXL1 with histones and DNA that are critical for restructuring the nucleosome and thus establishing specificity for H2AK119Ub. These results further provide a molecular explanation for how >50 mutations in BAP1 and ASXL1 found in cancer can dysregulate H2AK119Ub deubiquitination, providing insight into understanding cancer etiology.
PMID: 37556531
ISSN: 2375-2548
CID: 5594932

RNA polymerase drives ribonucleotide excision DNA repair in E. coli

Hao, Zhitai; Gowder, Manjunath; Proshkin, Sergey; Bharati, Binod K; Epshtein, Vitaly; Svetlov, Vladimir; Shamovsky, Ilya; Nudler, Evgeny
Ribonuclease HII (RNaseHII) is the principal enzyme that removes misincorporated ribonucleoside monophosphates (rNMPs) from genomic DNA. Here, we present structural, biochemical, and genetic evidence demonstrating that ribonucleotide excision repair (RER) is directly coupled to transcription. Affinity pull-downs and mass-spectrometry-assisted mapping of in cellulo inter-protein cross-linking reveal the majority of RNaseHII molecules interacting with RNA polymerase (RNAP) in E. coli. Cryoelectron microscopy structures of RNaseHII bound to RNAP during elongation, with and without the target rNMP substrate, show specific protein-protein interactions that define the transcription-coupled RER (TC-RER) complex in engaged and unengaged states. The weakening of RNAP-RNaseHII interactions compromises RER in vivo. The structure-functional data support a model where RNaseHII scans DNA in one dimension in search for rNMPs while "riding" the RNAP. We further demonstrate that TC-RER accounts for a significant fraction of repair events, thereby establishing RNAP as a surveillance "vehicle" for detecting the most frequently occurring replication errors.
PMID: 37196657
ISSN: 1097-4172
CID: 5503582

Control of transcription elongation and DNA repair by alarmone ppGpp

Weaver, Jacob W; Proshkin, Sergey; Duan, Wenqian; Epshtein, Vitaly; Gowder, Manjunath; Bharati, Binod K; Afanaseva, Elena; Mironov, Alexander; Serganov, Alexander; Nudler, Evgeny
Second messenger (p)ppGpp (collectively guanosine tetraphosphate and guanosine pentaphosphate) mediates bacterial adaptation to nutritional stress by modulating transcription initiation. More recently, ppGpp has been implicated in coupling transcription and DNA repair; however, the mechanism of ppGpp engagement remained elusive. Here we present structural, biochemical and genetic evidence that ppGpp controls Escherichia coli RNA polymerase (RNAP) during elongation via a specific site that is nonfunctional during initiation. Structure-guided mutagenesis renders the elongation (but not initiation) complex unresponsive to ppGpp and increases bacterial sensitivity to genotoxic agents and ultraviolet radiation. Thus, ppGpp binds RNAP at sites with distinct functions in initiation and elongation, with the latter being important for promoting DNA repair. Our data provide insights on the molecular mechanism of ppGpp-mediated adaptation during stress, and further highlight the intricate relationships between genome stability, stress responses and transcription.
PMID: 36997761
ISSN: 1545-9985
CID: 5463412

RNA polymerase and ppGpp deliver a one-two punch to antibiotics [Comment]

Rasouly, Aviram; Nudler, Evgeny
Mutation rates are elevated in response to sub-inhibitory concentrations of antibiotics. In this issue, Zhai et al.1 report a role for both ppGpp binding sites on RNAP in stress-induced mutagenesis.
PMID: 37084711
ISSN: 1097-4164
CID: 5464622