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1.
Cell ; 184(13): 3394-3409.e20, 2021 06 24.
Artículo en Inglés | MEDLINE | ID: mdl-34077752

RESUMEN

The human fetal immune system begins to develop early during gestation; however, factors responsible for fetal immune-priming remain elusive. We explored potential exposure to microbial agents in utero and their contribution toward activation of memory T cells in fetal tissues. We profiled microbes across fetal organs using 16S rRNA gene sequencing and detected low but consistent microbial signal in fetal gut, skin, placenta, and lungs in the 2nd trimester of gestation. We identified several live bacterial strains including Staphylococcus and Lactobacillus in fetal tissues, which induced in vitro activation of memory T cells in fetal mesenteric lymph node, supporting the role of microbial exposure in fetal immune-priming. Finally, using SEM and RNA-ISH, we visualized discrete localization of bacteria-like structures and eubacterial-RNA within 14th weeks fetal gut lumen. These findings indicate selective presence of live microbes in fetal organs during the 2nd trimester of gestation and have broader implications toward the establishment of immune competency and priming before birth.


Asunto(s)
Bacterias/metabolismo , Desarrollo Embrionario , Feto/citología , Feto/microbiología , Leucocitos/citología , Adulto , Bacterias/genética , Bacterias/ultraestructura , Proliferación Celular , Células Dendríticas/metabolismo , Femenino , Feto/ultraestructura , Tracto Gastrointestinal/embriología , Tracto Gastrointestinal/ultraestructura , Humanos , Memoria Inmunológica , Activación de Linfocitos/inmunología , Viabilidad Microbiana , Embarazo , Segundo Trimestre del Embarazo , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Reproducibilidad de los Resultados , Linfocitos T/citología
2.
Cell ; 180(5): 1002-1017.e31, 2020 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-32109417

RESUMEN

Genome-wide CRISPR screens enable systematic interrogation of gene function. However, guide RNA libraries are costly to synthesize, and their limited diversity compromises the sensitivity of CRISPR screens. Using the Streptococcus pyogenes CRISPR-Cas adaptation machinery, we developed CRISPR adaptation-mediated library manufacturing (CALM), which turns bacterial cells into "factories" for generating hundreds of thousands of crRNAs covering 95% of all targetable genomic sites. With an average gene targeted by more than 100 distinct crRNAs, these highly comprehensive CRISPRi libraries produced varying degrees of transcriptional repression critical for uncovering novel antibiotic resistance determinants. Furthermore, by iterating CRISPR adaptation, we rapidly generated dual-crRNA libraries representing more than 100,000 dual-gene perturbations. The polarized nature of spacer adaptation revealed the historical contingency in the stepwise acquisition of genetic perturbations leading to increasing antibiotic resistance. CALM circumvents the expense, labor, and time required for synthesis and cloning of gRNAs, allowing generation of CRISPRi libraries in wild-type bacteria refractory to routine genetic manipulation.


Asunto(s)
Sistemas CRISPR-Cas/genética , Genoma Bacteriano/genética , Biblioteca Genómica , Staphylococcus aureus/genética , Escherichia coli/genética , Humanos , ARN Bacteriano/genética , ARN Guía de Kinetoplastida/genética , Streptococcus pyogenes/genética
3.
Cell ; 179(1): 106-119.e16, 2019 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-31539491

RESUMEN

Genes are often transcribed by multiple RNA polymerases (RNAPs) at densities that can vary widely across genes and environmental conditions. Here, we provide in vitro and in vivo evidence for a built-in mechanism by which co-transcribing RNAPs display either collaborative or antagonistic dynamics over long distances (>2 kb) through transcription-induced DNA supercoiling. In Escherichia coli, when the promoter is active, co-transcribing RNAPs translocate faster than a single RNAP, but their average speed is not altered by large variations in promoter strength and thus RNAP density. Environmentally induced promoter repression reduces the elongation efficiency of already-loaded RNAPs, causing premature termination and quick synthesis arrest of no-longer-needed proteins. This negative effect appears independent of RNAP convoy formation and is abrogated by topoisomerase I activity. Antagonistic dynamics can also occur between RNAPs from divergently transcribed gene pairs. Our findings may be broadly applicable given that transcription on topologically constrained DNA is the norm across organisms.


Asunto(s)
ADN Bacteriano/genética , ADN Superhelicoidal/genética , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/genética , Transcripción Genética , ARN Polimerasas Dirigidas por ADN/química , Regulación Bacteriana de la Expresión Génica/genética , Glucosa/farmacología , Glicósidos/farmacología , Isopropil Tiogalactósido/farmacología , Cinética , Operón Lac/efectos de los fármacos , Operón Lac/genética , Plásmidos/genética , Regiones Promotoras Genéticas/genética , ARN Bacteriano/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Rifampin/farmacología
4.
Cell ; 170(4): 714-726.e10, 2017 Aug 10.
Artículo en Inglés | MEDLINE | ID: mdl-28757251

RESUMEN

Cas13a, a type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, degrades invasive RNAs targeted by CRISPR RNA (crRNA) and has potential applications in RNA technology. To understand how Cas13a is activated to cleave RNA, we have determined the crystal structure of Leptotrichia buccalis (Lbu) Cas13a bound to crRNA and its target RNA, as well as the cryo-EM structure of the LbuCas13a-crRNA complex. The crRNA-target RNA duplex binds in a positively charged central channel of the nuclease (NUC) lobe, and Cas13a protein and crRNA undergo a significant conformational change upon target RNA binding. The guide-target RNA duplex formation triggers HEPN1 domain to move toward HEPN2 domain, activating the HEPN catalytic site of Cas13a protein, which subsequently cleaves both single-stranded target and collateral RNAs in a non-specific manner. These findings reveal how Cas13a of type VI CRISPR-Cas systems defend against RNA phages and set the stage for its development as a tool for RNA manipulation.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Asociadas a CRISPR/química , Sistemas CRISPR-Cas , Leptotrichia/inmunología , Proteínas Bacterianas/ultraestructura , Secuencia de Bases , Proteínas Asociadas a CRISPR/ultraestructura , Leptotrichia/química , Leptotrichia/metabolismo , Leptotrichia/virología , Modelos Moleculares , Procesamiento Postranscripcional del ARN , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/ultraestructura , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/ultraestructura , ARN Viral/química , Difracción de Rayos X
5.
Mol Cell ; 84(18): 3482-3496.e7, 2024 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-39178862

RESUMEN

Binding of the bacterial Rho helicase to nascent transcripts triggers Rho-dependent transcription termination (RDTT) in response to cellular signals that modulate mRNA structure and accessibility of Rho utilization (Rut) sites. Despite the impact of temperature on RNA structure, RDTT was never linked to the bacterial response to temperature shifts. We show that Rho is a central player in the cold-shock response (CSR), challenging the current view that CSR is primarily a posttranscriptional program. We identify Rut sites in 5'-untranslated regions of key CSR genes/operons (cspA, cspB, cspG, and nsrR-rnr-yjfHI) that trigger premature RDTT at 37°C but not at 15°C. High concentrations of RNA chaperone CspA or nucleotide changes in the cspA mRNA leader reduce RDTT efficiency, revealing how RNA restructuring directs Rho to activate CSR genes during the cold shock and to silence them during cold acclimation. These findings establish a paradigm for how RNA thermosensors can modulate gene expression.


Asunto(s)
Regiones no Traducidas 5' , Respuesta al Choque por Frío , Proteínas de Escherichia coli , Escherichia coli , Regulación Bacteriana de la Expresión Génica , ARN Bacteriano , Factor Rho , Factor Rho/metabolismo , Factor Rho/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Respuesta al Choque por Frío/genética , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Terminación de la Transcripción Genética , Frío , Transcripción Genética , Operón , Proteínas y Péptidos de Choque por Frío
6.
Annu Rev Biochem ; 85: 319-47, 2016 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-27023849

RESUMEN

Transcript termination is essential for accurate gene expression and the removal of RNA polymerase (RNAP) at the ends of transcription units. In bacteria, two mechanisms are responsible for proper transcript termination: intrinsic termination and Rho-dependent termination. Intrinsic termination is mediated by signals directly encoded within the DNA template and nascent RNA, whereas Rho-dependent termination relies upon the adenosine triphosphate-dependent RNA translocase Rho, which binds nascent RNA and dissociates the elongation complex. Although significant progress has been made in understanding these pathways, fundamental details remain undetermined. Among those that remain unresolved are the existence of an inactivated intermediate in the intrinsic termination pathway, the role of Rho-RNAP interactions in Rho-dependent termination, and the mechanisms by which accessory factors and nucleoid-associated proteins affect termination. We describe current knowledge, discuss key outstanding questions, and highlight the importance of defining the structural rearrangements of RNAP that are involved in the two mechanisms of transcript termination.


Asunto(s)
ARN Polimerasas Dirigidas por ADN/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Factores de Elongación de Péptidos/genética , Factor Rho/genética , Factores de Transcripción/genética , Terminación de la Transcripción Genética , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Cinética , Modelos Moleculares , Conformación de Ácido Nucleico , Factores de Elongación de Péptidos/metabolismo , Unión Proteica , Transporte de Proteínas , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Factor Rho/metabolismo , Factores de Transcripción/metabolismo
7.
Genes Dev ; 38(13-14): 597-613, 2024 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-39111824

RESUMEN

Small RNAs base pair with and regulate mRNA translation and stability. For both bacterial small regulatory RNAs and eukaryotic microRNAs, association with partner proteins is critical for the stability and function of the regulatory RNAs. We review the mechanisms for degradation of these RNAs: displacement of the regulatory RNA from its protein partner (in bacteria) or destruction of the protein and its associated microRNAs (in eukaryotes). These mechanisms can allow specific destruction of a regulatory RNA via pairing with a decay trigger RNA or function as global off switches by disrupting the stability or function of the protein partner.


Asunto(s)
MicroARNs , Estabilidad del ARN , MicroARNs/metabolismo , MicroARNs/genética , Estabilidad del ARN/genética , Animales , Humanos , ARN Bacteriano/metabolismo , ARN Bacteriano/genética , Regulación de la Expresión Génica
8.
Cell ; 160(1-2): 228-40, 2015 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-25579683

RESUMEN

Quorum sensing is a cell-cell communication process that bacteria use to transition between individual and social lifestyles. In vibrios, homologous small RNAs called the Qrr sRNAs function at the center of quorum-sensing pathways. The Qrr sRNAs regulate multiple mRNA targets including those encoding the quorum-sensing regulatory components luxR, luxO, luxM, and aphA. We show that a representative Qrr, Qrr3, uses four distinct mechanisms to control its particular targets: the Qrr3 sRNA represses luxR through catalytic degradation, represses luxM through coupled degradation, represses luxO through sequestration, and activates aphA by revealing the ribosome binding site while the sRNA itself is degraded. Qrr3 forms different base-pairing interactions with each mRNA target, and the particular pairing strategy determines which regulatory mechanism occurs. Combined mathematical modeling and experiments show that the specific Qrr regulatory mechanism employed governs the potency, dynamics, and competition of target mRNA regulation, which in turn, defines the overall quorum-sensing response.


Asunto(s)
Percepción de Quorum , ARN Bacteriano/metabolismo , ARN Pequeño no Traducido/metabolismo , Vibrio/metabolismo , Secuencia de Bases , Escherichia coli/genética , Secuencias Invertidas Repetidas , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Pequeño no Traducido/química , ARN Pequeño no Traducido/genética , Vibrio/genética
9.
Nature ; 627(8003): 424-430, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38418874

RESUMEN

Mycobacterium tuberculosis (Mtb) is a bacterial pathogen that causes tuberculosis (TB), an infectious disease that is responsible for major health and economic costs worldwide1. Mtb encounters diverse environments during its life cycle and responds to these changes largely by reprogramming its transcriptional output2. However, the mechanisms of Mtb transcription and how they are regulated remain poorly understood. Here we use a sequencing method that simultaneously determines both termini of individual RNA molecules in bacterial cells3 to profile the Mtb transcriptome at high resolution. Unexpectedly, we find that most Mtb transcripts are incomplete, with their 5' ends aligned at transcription start sites and 3' ends located 200-500 nucleotides downstream. We show that these short RNAs are mainly associated with paused RNA polymerases (RNAPs) rather than being products of premature termination. We further show that the high propensity of Mtb RNAP to pause early in transcription relies on the binding of the σ-factor. Finally, we show that a translating ribosome promotes transcription elongation, revealing a potential role for transcription-translation coupling in controlling Mtb gene expression. In sum, our findings depict a mycobacterial transcriptome that prominently features incomplete transcripts resulting from RNAP pausing. We propose that the pausing phase constitutes an important transcriptional checkpoint in Mtb that allows the bacterium to adapt to environmental changes and could be exploited for TB therapeutics.


Asunto(s)
Regulación Bacteriana de la Expresión Génica , Mycobacterium tuberculosis , ARN Bacteriano , Transcriptoma , ARN Polimerasas Dirigidas por ADN/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , ARN Bacteriano/análisis , ARN Bacteriano/biosíntesis , ARN Bacteriano/genética , Transcriptoma/genética , Tuberculosis/microbiología , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Sitio de Iniciación de la Transcripción , Factor sigma/metabolismo , Ribosomas/metabolismo , Biosíntesis de Proteínas
10.
Mol Cell ; 82(3): 629-644.e4, 2022 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-35063132

RESUMEN

The envelope of Gram-negative bacteria is a vital barrier that must balance protection and nutrient uptake. Small RNAs are crucial regulators of the envelope composition and function. Here, using RIL-seq to capture the Hfq-mediated RNA-RNA interactome in Salmonella enterica, we discover envelope-related riboregulators, including OppX. We show that OppX acts as an RNA sponge of MicF sRNA, a prototypical porin repressor. OppX originates from the 5' UTR of oppABCDF, encoding the major inner-membrane oligopeptide transporter, and sequesters MicF's seed region to derepress the synthesis of the porin OmpF. Intriguingly, OppX operates as a true sponge, storing MicF in an inactive complex without affecting its levels or stability. Conservation of the opp-OppX-MicF-ompF axis in related bacteria suggests that it serves an important mechanism, adjusting envelope porosity to specific transport capacity. These data also highlight the resource value of this Salmonella RNA interactome, which will aid in unraveling RNA-centric regulation in enteric pathogens.


Asunto(s)
Regiones no Traducidas 5' , Membrana Celular/genética , Proteínas de Escherichia coli/genética , Proteína de Factor 1 del Huésped/genética , ARN Bacteriano/genética , Salmonella enterica/genética , Transporte Biológico , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteína de Factor 1 del Huésped/metabolismo , Interacciones Huésped-Patógeno , Permeabilidad , Porinas/genética , Porinas/metabolismo , ARN Bacteriano/metabolismo , RNA-Seq , Salmonella enterica/metabolismo , Salmonella enterica/patogenicidad
11.
Mol Cell ; 82(3): 616-628.e5, 2022 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-35051352

RESUMEN

Canonical CRISPR-Cas systems utilize RNA-guided nucleases for targeted cleavage of foreign nucleic acids, whereas some nuclease-deficient CRISPR-Cas complexes have been repurposed to direct the insertion of Tn7-like transposons. Here, we established a bioinformatic and experimental pipeline to comprehensively explore the diversity of Type I-F CRISPR-associated transposons. We report DNA integration for 20 systems and identify a highly active subset that exhibits complete orthogonality in transposon DNA mobilization. We reveal the modular nature of CRISPR-associated transposons by exploring the horizontal acquisition of targeting modules and by characterizing a system that encodes both a programmable, RNA-dependent pathway, and a fixed, RNA-independent pathway. Finally, we analyzed transposon-encoded cargo genes and found the striking presence of anti-phage defense systems, suggesting a role in transmitting innate immunity between bacteria. Collectively, this study substantially advances our biological understanding of CRISPR-associated transposon function and expands the suite of RNA-guided transposases for programmable, large-scale genome engineering.


Asunto(s)
Proteínas Bacterianas/genética , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Elementos Transponibles de ADN/genética , ADN Bacteriano/genética , Escherichia coli/genética , Evolución Molecular , Transposasas/genética , Proteínas Bacterianas/metabolismo , ADN Bacteriano/metabolismo , Escherichia coli/inmunología , Escherichia coli/metabolismo , Edición Génica , Regulación Bacteriana de la Expresión Génica , Variación Genética , Inmunidad Innata , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Transposasas/metabolismo
12.
Mol Cell ; 82(2): 404-419.e9, 2022 01 20.
Artículo en Inglés | MEDLINE | ID: mdl-34798057

RESUMEN

The epitranscriptome has emerged as a new fundamental layer of control of gene expression. Nevertheless, the determination of the transcriptome-wide occupancy and function of RNA modifications remains challenging. Here we have developed Rho-seq, an integrated pipeline detecting a range of modifications through differential modification-dependent rhodamine labeling. Using Rho-seq, we confirm that the reduction of uridine to dihydrouridine (D) by the Dus reductase enzymes targets tRNAs in E. coli and fission yeast. We find that the D modification is also present on fission yeast mRNAs, particularly those encoding cytoskeleton-related proteins, which is supported by large-scale proteome analyses and ribosome profiling. We show that the α-tubulin encoding mRNA nda2 undergoes Dus3-dependent dihydrouridylation, which affects its translation. The absence of the modification on nda2 mRNA strongly impacts meiotic chromosome segregation, resulting in low gamete viability. Applying Rho-seq to human cells revealed that tubulin mRNA dihydrouridylation is evolutionarily conserved.


Asunto(s)
Segregación Cromosómica , Escherichia coli/genética , Meiosis , Procesamiento Postranscripcional del ARN , ARN Bacteriano/genética , ARN de Hongos/genética , ARN Mensajero/genética , Schizosaccharomyces/genética , Uridina/metabolismo , Cromosomas Bacterianos , Cromosomas Fúngicos , Cromosomas Humanos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Evolución Molecular , Células HCT116 , Humanos , Oxidación-Reducción , ARN Bacteriano/metabolismo , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Análisis de Secuencia de ARN , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
13.
Nature ; 618(7964): 358-364, 2023 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-37225987

RESUMEN

The ability to switch between different lifestyles allows bacterial pathogens to thrive in diverse ecological niches1,2. However, a molecular understanding of their lifestyle changes within the human host is lacking. Here, by directly examining bacterial gene expression in human-derived samples, we discover a gene that orchestrates the transition between chronic and acute infection in the opportunistic pathogen Pseudomonas aeruginosa. The expression level of this gene, here named sicX, is the highest of the P. aeruginosa genes expressed in human chronic wound and cystic fibrosis infections, but it is expressed at extremely low levels during standard laboratory growth. We show that sicX encodes a small RNA that is strongly induced by low-oxygen conditions and post-transcriptionally regulates anaerobic ubiquinone biosynthesis. Deletion of sicX causes P. aeruginosa to switch from a chronic to an acute lifestyle in multiple mammalian models of infection. Notably, sicX is also a biomarker for this chronic-to-acute transition, as it is the most downregulated gene when a chronic infection is dispersed to cause acute septicaemia. This work solves a decades-old question regarding the molecular basis underlying the chronic-to-acute switch in P. aeruginosa and suggests oxygen as a primary environmental driver of acute lethality.


Asunto(s)
Enfermedad Aguda , Enfermedad Crónica , Genes Bacterianos , Oxígeno , Infecciones por Pseudomonas , Pseudomonas aeruginosa , ARN Bacteriano , Animales , Humanos , Oxígeno/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Infecciones por Pseudomonas/complicaciones , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/patología , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Fibrosis Quística/microbiología , Heridas y Lesiones/microbiología , Ubiquinona/biosíntesis , Anaerobiosis , Genes Bacterianos/genética , Sepsis/complicaciones , Sepsis/microbiología
14.
Nature ; 613(7945): 783-789, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36631609

RESUMEN

Efficient and accurate termination is required for gene transcription in all living organisms1,2. Cellular RNA polymerases in both bacteria and eukaryotes can terminate their transcription through a factor-independent termination pathway3,4-called intrinsic termination transcription in bacteria-in which RNA polymerase recognizes terminator sequences, stops nucleotide addition and releases nascent RNA spontaneously. Here we report a set of single-particle cryo-electron microscopy structures of Escherichia coli transcription intrinsic termination complexes representing key intermediate states of the event. The structures show how RNA polymerase pauses at terminator sequences, how the terminator RNA hairpin folds inside RNA polymerase, and how RNA polymerase rewinds the transcription bubble to release RNA and then DNA. These macromolecular snapshots define a structural mechanism for bacterial intrinsic termination and a pathway for RNA release and DNA collapse that is relevant for factor-independent termination by all RNA polymerases.


Asunto(s)
ADN Bacteriano , ARN Polimerasas Dirigidas por ADN , Escherichia coli , ARN Bacteriano , Terminación de la Transcripción Genética , Microscopía por Crioelectrón , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/ultraestructura , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/ultraestructura , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Bacteriano/ultraestructura , Regiones Terminadoras Genéticas/genética , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ADN Bacteriano/ultraestructura
15.
Mol Cell ; 81(1): 127-138.e4, 2021 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-33212019

RESUMEN

Riboswitches are thought generally to function by modulating transcription elongation or translation initiation. In rare instances, ligand binding to a riboswitch has been found to alter the rate of RNA degradation by directly stimulating or inhibiting nearby cleavage. Here, we show that guanidine-induced pseudoknot formation by the aptamer domain of a guanidine III riboswitch from Legionella pneumophila has a different effect, stabilizing mRNA by protecting distal cleavage sites en masse from ribonuclease attack. It does so by creating a coaxially base-paired obstacle that impedes scanning from a monophosphorylated 5' end to those sites by the regulatory endonuclease RNase E. Ligand binding by other riboswitch aptamers peripheral to the path traveled by RNase E does not inhibit distal cleavage. These findings reveal that a riboswitch aptamer can function independently of any overlapping expression platform to regulate gene expression by acting directly to prolong mRNA longevity in response to ligand binding.


Asunto(s)
Proteínas Bacterianas/metabolismo , Endorribonucleasas/metabolismo , Legionella pneumophila/metabolismo , Pliegue del ARN , ARN Bacteriano/metabolismo , Riboswitch , Proteínas Bacterianas/genética , Endorribonucleasas/genética , Legionella pneumophila/genética , ARN Bacteriano/genética
16.
Mol Cell ; 81(9): 1857-1858, 2021 05 06.
Artículo en Inglés | MEDLINE | ID: mdl-33961772

RESUMEN

We talk to Ewelina Malecka and Sarah Woodson about their paper, "Stepwise sRNA targeting of structured bacterial mRNAs leads to abortive annealing," who inspired them along their scientific paths, the research in Sarah's lab and the environment she looks to create, as well as Ewelina's advice for aspiring scientists.


Asunto(s)
Investigación Biomédica/historia , Genética/historia , ARN Bacteriano/historia , ARN Pequeño no Traducido/historia , Selección de Profesión , Regulación Bacteriana de la Expresión Génica , Historia del Siglo XX , Historia del Siglo XXI , Proteína de Factor 1 del Huésped/metabolismo , Humanos , Conformación de Ácido Nucleico , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , Relación Estructura-Actividad
17.
Mol Cell ; 81(14): 2901-2913.e5, 2021 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-34157309

RESUMEN

Polynucleotide phosphorylase (PNPase) is an ancient exoribonuclease conserved in the course of evolution and is found in species as diverse as bacteria and humans. Paradoxically, Escherichia coli PNPase can act not only as an RNA degrading enzyme but also by an unknown mechanism as a chaperone for small regulatory RNAs (sRNAs), with pleiotropic consequences for gene regulation. We present structures of the ternary assembly formed by PNPase, the RNA chaperone Hfq, and sRNA and show that this complex boosts sRNA stability in vitro. Comparison of structures for PNPase in RNA carrier and degradation modes reveals how the RNA is rerouted away from the active site through interactions with Hfq and the KH and S1 domains. Together, these data explain how PNPase is repurposed to protect sRNAs from cellular ribonucleases such as RNase E and could aid RNA presentation to facilitate regulatory actions on target genes.


Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Proteína de Factor 1 del Huésped/genética , Polirribonucleótido Nucleotidiltransferasa/genética , ARN Bacteriano/genética , Dominio Catalítico/genética , Endorribonucleasas/genética , Exorribonucleasas/genética , Regulación Bacteriana de la Expresión Génica/genética , Chaperonas Moleculares/genética , Estabilidad del ARN/genética , ARN Pequeño no Traducido/genética
18.
Mol Cell ; 81(1): 104-114.e6, 2021 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-33259811

RESUMEN

Aborted translation produces large ribosomal subunits obstructed with tRNA-linked nascent chains, which are substrates of ribosome-associated quality control (RQC). Bacterial RqcH, a widely conserved RQC factor, senses the obstruction and recruits tRNAAla(UGC) to modify nascent-chain C termini with a polyalanine degron. However, how RqcH and its eukaryotic homologs (Rqc2 and NEMF), despite their relatively simple architecture, synthesize such C-terminal tails in the absence of a small ribosomal subunit and mRNA has remained unknown. Here, we present cryoelectron microscopy (cryo-EM) structures of Bacillus subtilis RQC complexes representing different Ala tail synthesis steps. The structures explain how tRNAAla is selected via anticodon reading during recruitment to the A-site and uncover striking hinge-like movements in RqcH leading tRNAAla into a hybrid A/P-state associated with peptidyl-transfer. Finally, we provide structural, biochemical, and molecular genetic evidence identifying the Hsp15 homolog (encoded by rqcP) as a novel RQC component that completes the cycle by stabilizing the P-site tRNA conformation. Ala tailing thus follows mechanistic principles surprisingly similar to canonical translation elongation.


Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Extensión de la Cadena Peptídica de Translación , ARN Bacteriano/metabolismo , ARN de Transferencia de Alanina/metabolismo , Bacillus subtilis/ultraestructura , Proteínas Bacterianas/genética , Microscopía por Crioelectrón , ARN Bacteriano/genética , ARN de Transferencia de Alanina/genética
19.
Mol Cell ; 81(9): 1988-1999.e4, 2021 05 06.
Artículo en Inglés | MEDLINE | ID: mdl-33705712

RESUMEN

Bacterial small RNAs (sRNAs) regulate the expression of hundreds of transcripts via base pairing mediated by the Hfq chaperone protein. sRNAs and the mRNA sites they target are heterogeneous in sequence, length, and secondary structure. To understand how Hfq can flexibly match diverse sRNA and mRNA pairs, we developed a single-molecule Förster resonance energy transfer (smFRET) platform that visualizes the target search on timescales relevant in cells. Here we show that unfolding of target secondary structure on Hfq creates a kinetic energy barrier that determines whether target recognition succeeds or aborts before a stable anti-sense complex is achieved. Premature dissociation of the sRNA can be alleviated by strong RNA-Hfq interactions, explaining why sRNAs have different target recognition profiles. We propose that the diverse sequences and structures of Hfq substrates create an additional layer of information that tunes the efficiency and selectivity of non-coding RNA regulation in bacteria.


Asunto(s)
Escherichia coli K12/metabolismo , Regulación Bacteriana de la Expresión Génica , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , ARN Pequeño no Traducido/metabolismo , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Proteína de Factor 1 del Huésped/genética , Proteína de Factor 1 del Huésped/metabolismo , Cinética , Microscopía Fluorescente , Conformación de Ácido Nucleico , Estabilidad Proteica , Estructura Secundaria de Proteína , Desplegamiento Proteico , Estabilidad del ARN , ARN Bacteriano/genética , ARN Mensajero/genética , ARN Pequeño no Traducido/genética , Análisis de la Célula Individual , Relación Estructura-Actividad
20.
Mol Cell ; 81(2): 281-292.e8, 2021 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-33296676

RESUMEN

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/metabolismo
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