RESUMEN
Transcription elongation stalls at lesions in the DNA template1. For the DNA lesion to be repaired, the stalled transcription elongation complex (EC) has to be removed from the damaged site2. Here we show that translation, which is coupled to transcription in bacteria, actively dislodges stalled ECs from the damaged DNA template. By contrast, paused, but otherwise elongation-competent, ECs are not dislodged by the ribosome. Instead, they are helped back into processive elongation. We also show that the ribosome slows down when approaching paused, but not stalled, ECs. Our results indicate that coupled ribosomes functionally and kinetically discriminate between paused ECs and stalled ECs, ensuring the selective destruction of only the latter. This functional discrimination is controlled by the RNA polymerase's catalytic domain, the Trigger Loop. We show that the transcription-coupled DNA repair helicase UvrD, proposed to cause backtracking of stalled ECs3, does not interfere with ribosome-mediated dislodging. By contrast, the transcription-coupled DNA repair translocase Mfd4 acts synergistically with translation, and dislodges stalled ECs that were not destroyed by the ribosome. We also show that a coupled ribosome efficiently destroys misincorporated ECs that can cause conflicts with replication5. We propose that coupling to translation is an ancient and one of the main mechanisms of clearing non-functional ECs from the genome.
Asunto(s)
ARN Polimerasas Dirigidas por ADN , Escherichia coli , Biosíntesis de Proteínas , Transcripción Genética , Dominio Catalítico , ADN Helicasas/metabolismo , Reparación del ADN , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Cinética , Ribosomas/metabolismo , Moldes Genéticos , Elongación de la Transcripción Genética , Genoma BacterianoRESUMEN
Deciphering translation is of paramount importance for the understanding of many diseases, and antibiotics played a pivotal role in this endeavour. Blasticidin S (BlaS) targets translation by binding to the peptidyl transferase center of the large ribosomal subunit. Using biochemical, structural and cellular approaches, we show here that BlaS inhibits both translation elongation and termination in Mammalia. Bound to mammalian terminating ribosomes, BlaS distorts the 3'CCA tail of the P-site tRNA to a larger extent than previously reported for bacterial ribosomes, thus delaying both, peptide bond formation and peptidyl-tRNA hydrolysis. While BlaS does not inhibit stop codon recognition by the eukaryotic release factor 1 (eRF1), it interferes with eRF1's accommodation into the peptidyl transferase center and subsequent peptide release. In human cells, BlaS inhibits nonsense-mediated mRNA decay and, at subinhibitory concentrations, modulates translation dynamics at premature termination codons leading to enhanced protein production.
Asunto(s)
Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Terminación de la Cadena Péptídica Traduccional/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Microscopía por Crioelectrón , Células HeLa , Humanos , Degradación de ARNm Mediada por Codón sin Sentido/efectos de los fármacos , Nucleósidos/química , Nucleósidos/farmacología , Factores de Terminación de Péptidos/metabolismo , Péptidos/metabolismo , Inhibidores de la Síntesis de la Proteína/química , ARN Mensajero/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/química , Subunidades Ribosómicas Grandes de Eucariotas/efectos de los fármacos , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Ribosomas/metabolismoRESUMEN
In bacteria, the first two steps of gene expression-transcription and translation-are spatially and temporally coupled. Uncoupling may lead to the arrest of transcription through RNA polymerase backtracking, which interferes with replication forks, leading to DNA double-stranded breaks and genomic instability. How transcription-translation coupling mitigates these conflicts is unknown. Here we show that, unlike replication, translation is not inhibited by arrested transcription elongation complexes. Instead, the translating ribosome actively pushes RNA polymerase out of the backtracked state, thereby reactivating transcription. We show that the distance between the two machineries upon their contact on mRNA is smaller than previously thought, suggesting intimate interactions between them. However, this does not lead to the formation of a stable functional complex between the enzymes, as was once proposed. Our results reveal an active, energy-driven mechanism that reactivates backtracked elongation complexes and thus helps suppress their interference with replication.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/enzimología , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Transcripción Genética , Factores de Elongación Transcripcional/metabolismo , Secuencia de Bases , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/genética , ARN Mensajero/genética , Ribosomas/genética , Factores de Elongación Transcripcional/genéticaRESUMEN
The essential cell wall peptidoglycan is the target of several components of the innate immune system and its disruption results in lysis of invading bacteria. The pathogen Streptococcus pneumoniae produces a peptidoglycan N-acetylglucosamine deacetylase, PgdA, to modify the peptidoglycan structure. The activity of PgdA contributes to the bacteria's resistance to lysozyme, which is an important antimicrobial factor of the human innate immune system. In this study we report on the activity of PgdA against natural and artificial substrates. We have also established a virtual high-throughput screening and a new enzyme assay to search for compounds inhibiting PgdA. Two compounds with IC(50) values in the micromolar range have been identified and they could serve as leads for the search of inhibitors of PgdA, an important pneumococcal virulence factor.