RESUMEN
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.
Asunto(s)
Reparación del ADN , ARN Polimerasas Dirigidas por ADN , Escherichia coli , Microscopía por Crioelectrón , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/enzimología , Escherichia coli/metabolismo , Ribonucleótidos/metabolismoRESUMEN
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.
Asunto(s)
Antibacterianos , Sitios de Unión , ARN Polimerasas Dirigidas por ADN , Escherichia coli , Mutación , Rifampin , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Sitios de Unión/efectos de los fármacos , Sitios de Unión/genética , Roturas del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Farmacorresistencia Bacteriana/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Nucleótidos/deficiencia , Nucleótidos/metabolismo , Regiones Promotoras Genéticas , Rifampin/química , Rifampin/metabolismo , Rifampin/farmacología , Factores de Tiempo , Transcripción Genética/efectos de los fármacosRESUMEN
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.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Factores de Elongación de Péptidos/química , Factores de Transcripción/química , Terminación de la Transcripción Genética , Factores de Elongación Transcripcional/química , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Microscopía por Crioelectrón , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Modelos Moleculares , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
The molecular mechanisms responsible for axis establishment during non-embryonic processes remain elusive. The planarian flatworm is an ideal model organism to study body axis polarization and patterning in vivo. Here, we identified a homolog of the TBX2/3 in the planarian Dugesia japonica. RNA interference (RNAi) knockdown of TBX2/3 results in the ectopic formation of protrusions in the midline of the dorsal surface which shows an abnormal expression of midline and ventral cell markers. Additionally, the TBX2/3 RNAi animals also show the duplication of expression of the boundary marker at the lateral edge. Furthermore, TBX2/3 is expressed in muscle cells and co-expressed with bmp4. Inhibition of bone morphogenetic protein (BMP) signaling reduces the expression of TBX2/3 at the midline. These results suggest that TBX2/3 RNAi results in phenotypic characters caused by inhibition of the BMP signal, indicating that TBX2/3 is required for DV and ML patterning, and might be a downstream gene of BMP signaling.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Morfogénesis , Planarias/fisiología , Regeneración , Proteínas de Dominio T Box/metabolismo , Animales , Tipificación del Cuerpo , Proteínas Morfogenéticas Óseas/genética , Planarias/citología , Transducción de Señal , Proteínas de Dominio T Box/genéticaRESUMEN
Aminoacyl-tRNA synthetases are an ancient enzyme family that specifically charges tRNA molecules with cognate amino acids for protein synthesis. Glycyl-tRNA synthetase (GlyRS) is one of the most intriguing aminoacyl-tRNA synthetases due to its divergent quaternary structure and abnormal charging properties. In the past decade, mutations of human GlyRS (hGlyRS) were also found to be associated with Charcot-Marie-Tooth disease. However, the mechanisms of traditional and alternative functions of hGlyRS are poorly understood due to a lack of studies at the molecular basis. In this study we report crystal structures of wild type and mutant hGlyRS in complex with tRNA and with small substrates and describe the molecular details of enzymatic recognition of the key tRNA identity elements in the acceptor stem and the anticodon loop. The cocrystal structures suggest that insertions 1 and 3 work together with the active site in a cooperative manner to facilitate efficient substrate binding. Both the enzyme and tRNA molecules undergo significant conformational changes during glycylation. A working model of multiple conformations for hGlyRS catalysis is proposed based on the crystallographic and biochemical studies. This study provides insights into the catalytic pathway of hGlyRS and may also contribute to our understanding of Charcot-Marie-Tooth disease.
Asunto(s)
Glicina-ARNt Ligasa/química , ARN de Transferencia de Glicerina/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Dominio Catalítico/genética , Enfermedad de Charcot-Marie-Tooth/enzimología , Enfermedad de Charcot-Marie-Tooth/genética , Cristalografía por Rayos X , Glicina-ARNt Ligasa/genética , Glicina-ARNt Ligasa/metabolismo , Glicosilación , Células HeLa , Humanos , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Modelos Moleculares , Mutagénesis Insercional , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformación de Ácido Nucleico , Conformación Proteica , ARN de Transferencia de Glicerina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Thermus thermophilus/enzimología , Thermus thermophilus/genéticaRESUMEN
The 6-aminopurine ring of adenosine (A) can be deaminated to form the 6-oxopurine of inosine (I). Endonuclease Vs (EndoVs) are inosine-specific nucleases that cleave at the second phosphodiester bond 3' to inosine. EndoV proteins are highly conserved in all domains of life, but the bacterial and human enzymes seem to display distinct substrate preferences. While the bacterial enzymes exhibit high cleavage efficiency on various nucleic acid substrates, human EndoV (hEndoV) is most active towards ssRNA but is much less active towards other substrates. However, the structural basis of substrate recognition by hEndoV is not well understood. In this study, the 2.3â Å resolution crystal structure of hEndoV was determined and its unusual RNA-cleaving properties were investigated. The enzyme preserves the general `RNase H-like' structure, especially in the wedge motif, the metal-binding site and the hypoxanthine-binding pocket. hEndoV also features several extra insertions and a characteristic four-cysteine motif, in which Cys227 and Cys228, two cysteines that are highly conserved in higher eukaryotes, play important roles in catalysis. The structure presented here helps in understanding the substrate preference of hEndoV catalysis.
Asunto(s)
Desoxirribonucleasa (Dímero de Pirimidina)/química , Inosina/metabolismo , Ribonucleasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cristalografía por Rayos X , Desoxirribonucleasa (Dímero de Pirimidina)/genética , Desoxirribonucleasa (Dímero de Pirimidina)/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Adult stem cells (ASCs) are pluripotent cells with the capacity to self-renew and constantly replace lost cells due to physiological turnover or injury. Understanding the molecular mechanisms of the precise coordination of stem cell proliferation and proper cell fate decision is important to regeneration and organismal homeostasis. The planarian epidermis provides a highly tractable model to study ASC complex dynamic due to the distinct spatiotemporal differentiation stages during lineage development. Here, we identified the myosin regulatory light chain (MRLC) homologue in the Dugesia japonica transcriptome. We found high expression levels of MRLC in wound region during regeneration and also expressed in late epidermal progenitors as an essential regulator of the lineage from neoblasts to mature epidermal cells. We investigated the function of MRLC using in situ hybridization, real-time polymerase chain reaction and double fluorescent and uncovered the potential mechanism. Knockdown of MRLC leads to a remarkable increase in cell death, causes severe abnormalities during regeneration and homeostasis and eventually leads to animal death. The global decrease in epidermal cell in MRLC RNAi animals induces accelerated epidermal proliferation and differentiation. Additionally, we find that MRLC is co-expressed with cdc42 and acts cooperatively to control the epidermal lineage development by affecting cell death. Our results uncover an important role of MRLC, as an inhibitor of apoptosis, involves in epidermal development.
Asunto(s)
Planarias , Animales , Planarias/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Homeostasis/fisiología , Diferenciación Celular , ApoptosisRESUMEN
Rho is a hexameric bacterial RNA helicase, which became a paradigm of factor-dependent transcription termination. The broadly accepted ("textbook") model posits a series of steps, wherein Rho first binds C-rich Rho utilization (rut) sites on nascent RNA, uses its ATP-dependent translocase activity to catch up with RNA polymerase (RNAP), and either pulls the transcript from the elongation complex or pushes RNAP forward, thus terminating transcription. However, this appealingly simple mechano-chemical model lacks a biological realism and is increasingly at odds with genetic and biochemical data. Here, we summarize recent structural and biochemical studies that have advanced our understanding of molecular details of RNA recognition, termination signaling, and RNAP inactivation in Rho-dependent transcription termination, rebalancing the view in favor of an alternative "allosteric" mechanism. In the revised model, Rho binds RNAP early in elongation assisted by the cofactors NusA and NusG, forming a pre-termination complex (PTC). The formation of PTC allows Rho to continuously sample nascent transcripts for a termination signal, which subsequently traps the elongation complex in an inactive state prior to its dissociation.
Asunto(s)
Proteínas de Escherichia coli , Factor Rho , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Factor Rho/genética , Factor Rho/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
Tubgcp3/GCP3 (The centrosomal protein γ-tubulin complex protein 3) is a component of the γ-tubulin small complexes (γ-TuSCs) and γ-tubulin ring complexes (γ-TuRCs), which play critical roles in mitotic spindle formation during mitosis. However, its function in stem cell development has not been thoroughly elucidated. The planarian flatworm, which contains a large number of adult somatic stem cells (neoblasts), is a unique model to study stem cell lineage development in vivo. Here, we identified a homolog of Tubgcp3 in planarian Dugesia japonica, and found that Tubgcp3 is required for the maintenance of epidermal lineage. RNAi targeting Tubgcp3 resulted in tissue homeostasis and regeneration defect. Knockdown of Tubgcp3 reduced cell divisions and led to a loss of the mature epidermal cells. Our findings indicate that Tubgcp3 is a mitotic regulator and plays a crucial role in planarian epidermal differentiation.
Asunto(s)
Epidermis/fisiología , Proteínas Asociadas a Microtúbulos/genética , Animales , Diferenciación Celular , Proliferación Celular , Clonación Molecular , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Planarias , RegeneraciónRESUMEN
Mutations in the rifampicin (Rif)-binding site of RNA polymerase (RNAP) confer antibiotic resistance and often have global effects on transcription that compromise fitness and stress tolerance of resistant mutants. We suggested that the non-essential genome, through its impact on the bacterial transcription cycle, may represent an untapped source of targets for combination antimicrobial therapies. Using transposon sequencing, we carried out a genome-wide analysis of fitness cost in a clinically common rpoB H526Y mutant. We find that genes whose products enable increased transcription elongation rates compound the fitness costs of resistance whereas genes whose products function in cell wall synthesis and division mitigate it. We validate our findings by showing that the cell wall synthesis and division defects of rpoB H526Y result from an increased transcription elongation rate that is further exacerbated by the activity of the uracil salvage pathway and unresponsiveness of the mutant RNAP to the alarmone ppGpp. We applied our findings to identify drugs that inhibit more readily rpoB H526Y and other RifR alleles from the same phenotypic class. Thus, genome-wide analysis of fitness cost of antibiotic-resistant mutants should expedite the discovery of new combination therapies and delineate cellular pathways that underlie the molecular mechanisms of cost.