RESUMO
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.
Assuntos
RNA Polimerase II , RNA , Humanos , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA/genética , Transcrição Gênica , RNA Polimerases Dirigidas por DNA/genéticaRESUMO
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.
Assuntos
Antibacterianos , Sítios de Ligação , RNA Polimerases Dirigidas por DNA , Escherichia coli , Mutação , Rifampina , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Quebras de DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Nucleotídeos/deficiência , Nucleotídeos/metabolismo , Regiões Promotoras Genéticas , Rifampina/química , Rifampina/metabolismo , Rifampina/farmacologia , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacosRESUMO
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.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Guanosina Tetrafosfato/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Regulação Bacteriana da Expressão GênicaRESUMO
RNA polymerase II (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 is unknown. Consequently, we developed a novel method to directly sequence the extruded, "backtracked" 3' RNA. Our data shows that pol II slides backwards more than 20 nucleotides in human cells and can persist in this backtracked state. Persistent backtracking mainly occurs where 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 has the potential to affect diverse gene expression programs.
Assuntos
Antibacterianos , Bactérias , Farmacorresistência Bacteriana , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/enzimologia , Testes de Sensibilidade Microbiana , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismoRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Rifampina/farmacologia , Bactérias/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Farmacorresistência Bacteriana , Genoma Bacteriano , Mutação , Transcrição GênicaRESUMO
Prokaryotes and eukaryotes alike endogenously generate the gaseous molecule hydrogen sulfide (H2S). Bacterial H2S acts as a cytoprotectant against antibiotics-induced stress and promotes redox homeostasis. In E. coli, endogenous H2S production is primarily dependent on 3-mercaptopyruvate sulfurtransferase (3MST), encoded by mstA. Here, we show that cells lacking 3MST acquire a phenotypic suppressor mutation resulting in compensatory H2S production and tolerance to antibiotics and oxidative stress. Using whole genome sequencing, we identified a non-synonymous mutation within an uncharacterized LacI-type transcription factor, ycjW. We then mapped regulatory targets of YcjW and discovered it controls the expression of carbohydrate metabolic genes and thiosulfate sulfurtransferase PspE. Induction of pspE expression in the suppressor strain provides an alternative mechanism for H2S biosynthesis. Our results reveal a complex interaction between carbohydrate metabolism and H2S production in bacteria and the role, a hitherto uncharacterized transcription factor, YcjW, plays in linking the two.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Sulfeto de Hidrogênio/metabolismo , Fatores de Transcrição/metabolismo , Substituição de Aminoácidos , Antibacterianos/farmacologia , Mapeamento Cromossômico , DNA Bacteriano , Proteínas de Ligação a DNA/genética , Dissacarídeos/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/fisiologia , Ligação Proteica , RNA Mensageiro , Regulon , Fatores de Transcrição/genéticaRESUMO
In bacteria, mRNA transcription and translation are coupled to coordinate optimal gene expression and maintain genome stability. Coupling is thought to involve direct interactions between RNA polymerase (RNAP) and the translational machinery. We present cryo-EM structures of E. coli RNAP core bound to the small ribosomal 30S subunit. The complex is stable under cell-like ionic conditions, consistent with functional interaction between RNAP and the 30S subunit. The RNA exit tunnel of RNAP aligns with the Shine-Dalgarno-binding site of the 30S subunit. Ribosomal protein S1 forms a wall of the tunnel between RNAP and the 30S subunit, consistent with its role in directing mRNAs onto the ribosome. The nucleic-acid-binding cleft of RNAP samples distinct conformations, suggesting different functional states during transcription-translation coupling. The architecture of the 30Sâ¢RNAP complex provides a structural basis for co-localization of the transcriptional and translational machineries, and inform future mechanistic studies of coupled transcription and translation.
Assuntos
RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Microscopia Crioeletrônica , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismoRESUMO
Nucleotide excision repair (NER) is the key DNA repair system that eliminates the majority of DNA helix-distorting lesions. RNA polymerase (RNAP) expedites the recognition of DNA damage by NER components via transcription-coupled DNA repair (TCR). In bacteria, a modified nucleotide ppGpp ('magic spot') is a pleiotropic second messenger that mediates the response to nutrient deficiencies by altering the initiation properties of RNAP. In this review, we discuss newly elucidated roles of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) in transcription elongation that couple this alarmone to DNA damage repair and maintenance.
Assuntos
Reparo do DNA/genética , RNA Polimerases Dirigidas por DNA/genética , Transcrição Gênica , DNA/genética , Dano ao DNA/genéticaRESUMO
In 1943, Luria and Delbrück used a phage-resistance assay to establish spontaneous mutation as a driving force of microbial diversity. Mutation rates are still studied using such assays, but these can only be used to examine the small minority of mutations conferring survival in a particular condition. Newer approaches, such as long-term evolution followed by whole-genome sequencing, may be skewed by mutational 'hot' or 'cold' spots. Both approaches are affected by numerous caveats. Here we devise a method, maximum-depth sequencing (MDS), to detect extremely rare variants in a population of cells through error-corrected, high-throughput sequencing. We directly measure locus-specific mutation rates in Escherichia coli and show that they vary across the genome by at least an order of magnitude. Our data suggest that certain types of nucleotide misincorporation occur 10(4)-fold more frequently than the basal rate of mutations, but are repaired in vivo. Our data also suggest specific mechanisms of antibiotic-induced mutagenesis, including downregulation of mismatch repair via oxidative stress, transcriptionreplication conflicts, and, in the case of fluoroquinolones, direct damage to DNA.
Assuntos
Escherichia coli/genética , Evolução Molecular , Variação Genética/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutagênese/genética , Taxa de Mutação , Antibacterianos/farmacologia , Dano ao DNA/genética , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/genética , Replicação do DNA/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Fluoroquinolonas/farmacologia , Loci Gênicos/efeitos dos fármacos , Loci Gênicos/genética , Variação Genética/efeitos dos fármacos , Genoma Bacteriano/efeitos dos fármacos , Genoma Bacteriano/genética , Mutação INDEL/genética , Mutagênese/efeitos dos fármacos , Nucleotídeos/genética , Nucleotídeos/metabolismo , Estresse Oxidativo/genética , Transcrição Gênica/genéticaRESUMO
The involvement of type I chaperonins in bacterial and organellar protein folding has been well-documented. In E. coli and mitochondria, these ubiquitous and highly conserved proteins form chaperonin oligomers of identical 60 kDa subunits (cpn60), while in chloroplasts, two distinct cpn60 α and ß subunit types co-exist together. The primary sequence of α and ß subunits is ~50% identical, similar to their respective homologies to the bacterial GroEL. Moreover, the A. thaliana genome contains two α and four ß genes. The functional significance of this variability in plant chaperonin proteins has not yet been elucidated. In order to gain insight into the functional variety of the chloroplast chaperonin family members, we reconstituted ß homo-oligomers from A. thaliana following their expression in bacteria and subjected them to a structure-function analysis. Our results show for the first time, that A. thaliana ß homo-oligomers can function in vitro with authentic chloroplast co-chaperonins (ch-cpn10 and ch-cpn20). We also show that oligomers made up of different ß subunit types have unique properties and different preferences for co-chaperonin partners. We propose that chloroplasts may contain active ß homo-oligomers in addition to hetero-oligomers, possibly reflecting a variety of cellular roles.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Chaperoninas do Grupo I/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Genoma de Planta , Chaperoninas do Grupo I/química , Chaperoninas do Grupo I/genética , Dobramento de Proteína , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologiaRESUMO
The Escherichia coli yjbEFGH operon, encoding genes involved in exopolysaccharide production, has previously been shown to be induced by osmotic stress and to be negatively regulated by σ(38). Promoter analysis suggested that like most E. coli genes, its transcription is driven by the housekeeping sigma factor σ(70). Indeed, manipulation of any of the other five alternative sigma factors did not affect its induction by osmotic stress. Surprisingly, when assayed in a strain expressing low levels of σ(70), yjbEFGH induction in response to osmotic stress was higher than in a strain expressing normal levels of σ(70). Similar phenomena were observed in the σ(70)-driven promoters of sulA, uvrA, recA, fecI, entC and lacZ, the transcription of which is directly controlled by a repressor protein (LexA, Fur and LacI), but not in promoters of the housekeeping genes ftsA and ftsY, or in σ(38)-driven treA promoter. Since transcription factors are generally present in the cell in low numbers, we hypothesize that a decrease in σ(70), that drives the expression of lexA, fur and lacI as well, further diminishes their number in the cell and thus de-represses the induction of genes which are subjected to their repression.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Regulação para Baixo , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Fator sigma/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Óperon , Fator sigma/metabolismoRESUMO
The highly conserved bacterial ybeY gene is a heat shock gene whose function is not fully understood. Previously, we showed that the YbeY protein is involved in protein synthesis, as Escherichia coli mutants with ybeY deleted exhibit severe translational defects in vivo. Here we show that the in vitro activity of the translation machinery of ybeY deletion mutants is significantly lower than that of the wild type. We also show that the lower efficiency of the translation machinery is due to impaired 30S small ribosomal subunits.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Metaloproteínas/metabolismo , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Immunoblotting , Metaloproteínas/genética , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , Subunidades Ribossômicas Menores de Bactérias/genéticaRESUMO
Escherichia coli can grow at a broad temperature range, from less than 20 degrees C up to 45 degrees C. An increase in temperature results in a major physiological change, as enzymes work faster but, on the other hand, proteins tend to unfold. Therefore, a shift-up in temperature results in the induction of several regulatory response mechanisms aimed at restoring balanced growth at the new temperature. One important mechanism involves temperature-dependent proteolysis, which constitutes a fast response to temperature shift-ups. Here we discuss the effect of proteolysis on protein synthesis, and the heat shock response.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Temperatura , Proteases Dependentes de ATP/metabolismo , Biocatálise , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/genética , Metionina/biossíntese , Fator sigma/metabolismo , Estresse FisiológicoRESUMO
Bacteria respond to shift-up in temperature by activating the heat shock response - induction of a large number of heat shock genes. This response is essential for adaptation to the higher temperature and for acquiring thermotolerance. One unique feature of the heat shock response is its transient nature - shortly after the induction, the rate of synthesis of heat shock proteins decreases, even if the temperature remains high. Here we show that this shutoff is due to a decrease in the transcript stability of heat shock genes. We further show that the modulation of stability of mRNAs of heat shock genes is maintained by the cold shock protein C - CspC - previously shown to affect transcript stability of specific genes. Upon shifts to higher temperatures the level of this protein decreases due to proteolysis and aggregation, leading to a reduced stability of mRNAs of heat shock genes. The temperature-dependent modulation of transcript stability of heat shock genes constitutes a novel control of the bacterial response to temperature changes.
Assuntos
Adaptação Fisiológica/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/genética , Resposta ao Choque Térmico/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Temperatura Alta , Estabilidade de RNA , RNA Mensageiro/metabolismo , Fator sigma/genética , Fator sigma/metabolismoRESUMO
It is widely accepted that the heat shock response is critical for quality control of mature proteins. This function is carried out mainly by chaperones and proteases. Recently, a new group of conserved heat shock proteins essential for growth at high temperature has been characterized. These proteins are involved in regulating and maintaining efficient translation under heat shock.
Assuntos
Escherichia coli/metabolismo , Resposta ao Choque Térmico , Biossíntese de Proteínas , Adaptação Fisiológica/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Redes e Vias Metabólicas , Chaperonas Moleculares/fisiologia , Peptídeo Hidrolases/fisiologiaRESUMO
Here we provide evidence that YbeY, a conserved heat shock protein with unknown function, is involved in the translation process. ybeY deletion mutants are temperature sensitive and have a significantly reduced thermotolerance. Nonetheless, there appears to be no damage of the protein quality control of mature polypeptides, as the levels of chaperones and proteases are normal and there is no accumulation of aggregates. Rather, the mutation results in a significant reduction in the level of polysomes, and upon a shift to a restrictive temperature (42 degrees C), there is an immediate and severe slowdown of translation. Taken together, the data indicate that YbeY is an important factor for bacterial translation even at 37 degrees C but becomes essential at high temperatures.
Assuntos
Proteínas de Escherichia coli/fisiologia , Escherichia coli/fisiologia , Metaloproteínas/fisiologia , Biossíntese de Proteínas , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Deleção de Genes , Temperatura Alta , Metaloproteínas/genética , Polirribossomos/metabolismoRESUMO
Response to changes in light conditions involves a variety of receptors that can modulate gene expression, enzyme activity and/or motility. For the study of light-regulated effects of Agrobacterium tumefaciens, we used a global analysis approach - proteomics - and compared the protein patterns of dark- and light-grown bacteria. These analyses revealed a significant reduction of FlaA and FlaB - proteins of the flagellum - when the cells were grown in light. The light effect was confirmed by SDS-PAGE with isolated flagella. Quantitative PCR experiments showed a 10-fold increase of the transcription level of flaA, flaB and flaC within 20 min after the transfer from light to darkness. Electron microscopy revealed that these molecular events result in a light-induced reduction of the number of flagella per cell. These changes have major physiological consequences regarding motility, which is considerably reduced with exposure to light. The inhibitory effect of light on the motility is not unique to A. tumefaciens and was also seen in other species of the Rhizobiaceae. Previous studies suggested that the flagella function is significant for bacteria-plant interactions and bacterial virulence. In our studies, light reduced the attachment of A. tumefaciens to tomato roots and the virulence of the bacteria in a cucumber infection assay.