RESUMO
The ability of activated progenitor T cells to self-renew while producing differentiated effector cell descendants may underlie immunological memory and persistent responses to ongoing infection. The nature of stem-like T cells responding to cancer and during treatment with immunotherapy is not clear. The subcellular organization of dividing progenitor CD8+ T cells from mice challenged with syngeneic tumors is examined here. Three-dimensional microscopy reveals an activating hub composed of polarized CD3, CD28, and phosphatidylinositol 3-kinase (PI3K) activity at the putative immunological synapse with an inhibitory hub composed of polarized PD-1 and CD73 at the opposite pole of mitotic blasts. Progenitor T cells from untreated and inhibitory checkpoint blockade-treated mice yield a differentiated TCF1- daughter cell, which inherits the PI3K activation hub, alongside a discordantly fated, self-renewing TCF1+ sister cell. Dynamic organization of opposite activating and inhibitory signaling poles in mitotic lymphocytes may account for the enigmatic durability of specific immunity.
Assuntos
Linfócitos T CD8-Positivos , Fosfatidilinositol 3-Quinases , Camundongos , Animais , Diferenciação Celular , Células-Tronco , Transdução de SinaisRESUMO
Treatment with immune checkpoint blockade (ICB) often fails to elicit durable antitumor immunity. Recent studies suggest that ICB does not restore potency to terminally dysfunctional T cells, but instead drives proliferation and differentiation of self-renewing progenitor T cells into fresh, effector-like T cells. Antitumor immunity catalyzed by ICB is characterized by mobilization of antitumor T cells in systemic circulation and tumor. To address whether abundance of self-renewing T cells in blood is associated with immunotherapy response, we used flow cytometry of peripheral blood from a cohort of patients with metastatic non-small cell lung cancer (NSCLC) treated with ICB. At baseline, expression of T-cell factor 1 (TCF1), a marker of self-renewing T cells, was detected at higher frequency in effector-memory (CCR7-) CD8+ T cells from patients who experienced durable clinical benefit compared to those with primary resistance to ICB. On-treatment blood samples from patients benefiting from ICB also exhibited a greater frequency of TCF1+CCR7-CD8+ T cells and higher proportions of TCF1 expression in treatment-expanded PD-1+CCR7-CD8+ T cells. The observed correlation of TCF1 frequency in CCR7-CD8+ T cells and response to ICB suggests that broader examination of self-renewing T-cell abundance in blood will determine its potential as a noninvasive, predictive biomarker of response and resistance to immunotherapy.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Receptores CCR7 , Linfócitos T CD8-Positivos , ImunoterapiaRESUMO
It has been known for more than 50 years that transcription and translation are physically coupled in bacteria, but whether or not this coupling may be mediated by the two-domain protein N-utilization substance (Nus) G in Escherichia coli is still heavily debated. Here, we combine integrative structural biology and functional analyses to provide conclusive evidence that NusG can physically link transcription with translation by contacting both RNA polymerase and the ribosome. We present a cryo-electron microscopy structure of a NusG:70S ribosome complex and nuclear magnetic resonance spectroscopy data revealing simultaneous binding of NusG to RNAP and the intact 70S ribosome, providing the first direct structural evidence for NusG-mediated coupling. Furthermore, in vivo reporter assays show that recruitment of NusG occurs late in transcription and strongly depends on translation. Thus, our data suggest that coupling occurs initially via direct RNAP:ribosome contacts and is then mediated by NusG.
RESUMO
This article will review our current understanding of transcription elongation and termination in E. coli. We discuss why transcription elongation complexes pause at certain template sites and how auxiliary host and phage transcription factors affect elongation and termination. The connection between translation and transcription elongation is described. Finally we present an overview indicating where progress has been made and where it has not.
Assuntos
Regulação Bacteriana da Expressão Gênica , Elongação da Transcrição Genética , Terminação da Transcrição Genética , Sequência de Aminoácidos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Dados de Sequência Molecular , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
We have screened the entire KEIO collection of 3,985 single-gene knockouts in Escherichia coli for increased susceptibility or resistance to the antibiotic bicyclomycin (BCM), a potent inhibitor of the transcription termination factor Rho. We also compared the results to those of a recent study we conducted with a large set of antibiotics (A. Liu et al., Antimicrob. Agents Chemother. 54:1393-1403, 2010). We find that deletions of many different types of genes increase sensitivity to BCM. Some of these are involved in multidrug sensitivity/resistance, whereas others are specific for BCM. Mutations in a number of DNA recombination and repair genes increase BCM sensitivity, indicating that DNA damage leading to single- and double-strand breaks is a downstream effect of Rho inhibition. MDS42, which is deleted for all cryptic prophages and insertion elements (G. Posfai et al., Science 312:1044-1046, 2006), or W3102 deleted for the rac prophage-encoded kil gene, are partially resistant to BCM (C. J. Cardinale et al., Science 230:935-938, 2008). Deletion of cryptic prophages also overcomes the increased BCM sensitivity in some but not all mutants examined here. Deletion of the hns gene renders the cell more sensitive to BCM even in the Δkil or MDS42 background. This suggests that BCM activates additional modes of cell death independent of Kil and that these could provide a target to potentiate BCM killing.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Fator Rho/antagonistas & inibidores , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/fisiologia , Prófagos/genéticaRESUMO
DNA replication fork movement is impeded by collisions with transcription elongation complexes (TEC). We propose that a critical function of transcription termination factors is to prevent TEC from blocking DNA replication and inducing replication fork arrest, one consequence of which is DNA double-strand breaks. We show that inhibition of Rho-dependent transcription termination by bicyclomycin in Escherichia coli induced double-strand breaks. Cells deleted for Rho-cofactors nusA and nusG were hypersensitive to bicyclomycin, and had extensive chromosome fragmentation even in the absence of the drug. An RNA polymerase mutation that destabilizes TEC (rpoB*35) increased bicyclomycin resistance >40-fold. Double-strand break formation depended on DNA replication, and can be explained by replication fork collapse. Deleting recombination genes required for replication fork repair (recB and ruvC) increased sensitivity to bicyclomycin, as did loss of the replication fork reloading helicases rep and priA. We propose that Rho responds to a translocating replisome by releasing obstructing TEC.
Assuntos
Cromossomos/ultraestrutura , Escherichia coli/genética , Transcrição Gênica , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Cromossomos/efeitos dos fármacos , Análise Mutacional de DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/genética , RNA Polimerases Dirigidas por DNA/genética , Proteínas de Escherichia coli/genética , Modelos Genéticos , Mutação , Fatores de Alongamento de Peptídeos/genética , Fatores de Transcrição/genética , Fatores de Elongação da Transcrição , Raios UltravioletaRESUMO
The RNA sequences boxA, boxB and boxC constitute the nut regions of phage lambda. They nucleate the formation of a termination-resistant RNA polymerase complex on the lambda chromosome. The complex includes E. coli proteins NusA, NusB, NusG and NusE, and the lambda N protein. A complex that includes the Nus proteins and other factors forms at the rrn leader. Whereas RNA-binding by NusB and NusE has been described in quantitative terms, the interaction of NusA with these RNA sequences is less defined. Isotropic as well as anisotropic fluorescence equilibrium titrations show that NusA binds only the nut spacer sequence between boxA and boxB. Thus, nutR boxA5-spacer, nutR boxA16-spacer and nutR boxA69-spacer retain NusA binding, whereas a spacer mutation eliminates complex formation. The affinity of NusA for nutL is 50% higher than for nutR. In contrast, rrn boxA, which includes an additional U residue, binds NusA in the absence of spacer. The K(d) values obtained for rrn boxA and rrn boxA-spacer are 19-fold and 8-fold lower, respectively, than those for nutR boxA-spacer. These differences may explain why lambda requires an additional protein, lambda N, to suppress termination. Knowledge of the different affinities now describes the assembly of the anti-termination complex in quantitative terms.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Regiões 5' não Traduzidas , Proteínas de Bactérias/química , Bacteriófago lambda/genética , Sequência de Bases , Sítios de Ligação , Proteínas de Escherichia coli/química , Genes de RNAr , Dados de Sequência Molecular , Óperon , Fatores de Alongamento de Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/química , Fatores de Transcrição/química , Fatores de Elongação da TranscriçãoRESUMO
Transcription of the bacterial genome by the RNA polymerase must terminate at specific points. Transcription can be terminated by Rho factor, an essential protein in enterobacteria. We used the antibiotic bicyclomycin, which inhibits Rho, to assess its role on a genome-wide scale. Rho is revealed as a global regulator of gene expression that matches Escherichia coli transcription to translational needs. We also found that genes in E. coli that are most repressed by Rho are prophages and other horizontally acquired portions of the genome. Elimination of these foreign DNA elements increases resistance to bicyclomycin. Although rho remains essential, such reduced-genome bacteria no longer require Rho cofactors NusA and NusG. Deletion of the cryptic rac prophage in wild-type E. coli increases bicyclomycin resistance and permits deletion of nusG. Thus, Rho termination, supported by NusA and NusG, is required to suppress the toxic activity of foreign genes.
Assuntos
Escherichia coli O157/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Inativação Gênica , Fatores de Alongamento de Peptídeos/metabolismo , Prófagos/genética , Fator Rho/metabolismo , Fatores de Transcrição/metabolismo , Antibacterianos/farmacologia , Bacteriófago lambda/genética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Colífagos/genética , DNA Intergênico , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Análise de Sequência com Séries de Oligonucleotídeos , Proteoma , Fator Rho/antagonistas & inibidores , Transcrição Gênica , Fatores de Elongação da TranscriçãoRESUMO
The phage HK022 Nun protein excludes phage lambda by binding nascent lambda pL and pR transcripts at nutL and nutR, respectively, and inducing transcription termination just downstream of these sites. Termination is more efficient at nutL than at nutR. One difference between nutL and nutR is the presence of RNase III processing sites (rIII) located immediately promoter distal to lambda nutL. We found that deletion of rIII dramatically reduced Nun transcription arrest in vitro but had little effect on termination in vivo. However, consistent with the in vitro results, overexpression of a transcript carrying nutL and rIII efficiently titrated Nun, allowing lambda to grow on a strain that expressed Nun, whereas a transcript carrying only nutL or nutL-rIII with nucleotides 97 to 141 deleted was ineffective. Rnc70, an RNase III mutant that binds but does not cleave rIII, also prevented Nun-mediated lambda exclusion. We propose that rIII enhances the on-rate of Nun at nutL, stimulating Nun-mediated arrest in vitro. We have shown that a specific element in rIII, i.e., box C (G89GUGUGUG), strongly enhances arrest on rIII+ templates. Nun-rIII interactions do not stimulate Nun termination in vivo, presumably because formation of the Nun-nutL complex is normally not rate-limiting in the cell. In contrast to Nun, N is not occluded by Rnc70 and is not efficiently titrated by a nutL-rIII transcript.
Assuntos
Bacteriófago lambda/genética , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Ribonuclease III/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas Virais/metabolismo , Bacteriófago lambda/metabolismo , Sequência de Bases , Sítios de Ligação , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Deleção de Sequência , Regiões Terminadoras Genéticas , Fatores de Transcrição/genética , Proteínas Virais/genéticaRESUMO
The 109 amino acid residue Nun protein expressed from prophage HK022 excludes superinfecting phage lambda by arresting transcription on the lambda chromosome near the lambdanut sites. In vitro, the Nun N terminus binds to nascent lambdanutRNA, whereas the C terminus interacts with RNA polymerase and DNA template. Escherichia coli host factors, NusA, NusB, NusE (S10), and NusG, stimulate Nun-arrest. NusA binds the Nun C terminus and enhances formation of the Nun-nutRNA complex. Because of these in vitro activities of NusA, and since a nusA mutation (nusAE136K) blocked Nun in vivo, we assumed that NusA was required for Nun activity. However, using a nusAts strain, we find that NusA is required for termination at nutR but not at nutL. Furthermore, nusAE136K is dominant to nusA(+) for Nun-arrest, both in vitro and in vivo. NusAE136K shows increased affinity for Nun and, unlike NusA(+), can readily be recovered in a ternary complex with Nun and nutRNA. We propose NusAE136K suppresses Nun-arrest when it is a component of the transcription elongation complex, perhaps, in part, by blocking interactions between the Nun C terminus and RNA polymerase and DNA. We also find that in contrast to Nun-arrest, antitermination by lambda N requires NusA.
Assuntos
Bacteriófago HK022/metabolismo , Proteínas de Escherichia coli/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Regiões Terminadoras Genéticas , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas Virais/metabolismo , Bacteriófago HK022/genética , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/genética , Substâncias Macromoleculares , Modelos Genéticos , Mutação , Fatores de Alongamento de Peptídeos/genética , Fatores de Transcrição/genética , Fatores de Elongação da Transcrição , Proteínas Virais/genéticaRESUMO
Phage HK022 Nun protein excludes phage lambda by binding nascent lambda-nut RNA and inducing termination and transcript release. In contrast, in a purified in vitro system, Nun arrests transcription on lambdaDNA templates without dissociation of the transcription elongation complex (TEC). Our evidence indicates that transcription-repair coupling factor (Mfd) frees Nun-arrested RNA polymerase. The activity of Nun is enhanced in an mfd-null mutant, consistent with prolonged association of Nun with the TEC. Furthermore, expression of lambda nut RNA in the mfd mutant titrates Nun, allowing superinfecting lambda to form plaques. Finally, addition of Mfd releases a Nun-arrested transcription complex in vitro.