RESUMO
Drug-resistant bacteria are emerging as a global threat, despite frequently being less fit than their drug-susceptible ancestors1-8. Here we sought to define the mechanisms that drive or buffer the fitness cost of rifampicin resistance (RifR) in the bacterial pathogen Mycobacterium tuberculosis (Mtb). Rifampicin inhibits RNA polymerase (RNAP) and is a cornerstone of modern short-course tuberculosis therapy9,10. However, RifR Mtb accounts for one-quarter of all deaths due to drug-resistant bacteria11,12. We took a comparative functional genomics approach to define processes that are differentially vulnerable to CRISPR interference (CRISPRi) inhibition in RifR Mtb. Among other hits, we found that the universally conserved transcription factor NusG is crucial for the fitness of RifR Mtb. In contrast to its role in Escherichia coli, Mtb NusG has an essential RNAP pro-pausing function mediated by distinct contacts with RNAP and the DNA13. We find this pro-pausing NusG-RNAP interface to be under positive selection in clinical RifR Mtb isolates. Mutations in the NusG-RNAP interface reduce pro-pausing activity and increase fitness of RifR Mtb. Collectively, these results define excessive RNAP pausing as a molecular mechanism that drives the fitness cost of RifR in Mtb, identify a new mechanism of compensation to overcome this cost, suggest rational approaches to exacerbate the fitness cost, and, more broadly, could inform new therapeutic approaches to develop drug combinations to slow the evolution of RifR in Mtb.
Assuntos
Proteínas de Bactérias , Farmacorresistência Bacteriana , Evolução Molecular , Aptidão Genética , Mycobacterium tuberculosis , Rifampina , Humanos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência Conservada , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genômica , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Rifampina/farmacologia , Rifampina/uso terapêutico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
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.
Assuntos
Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis , RNA Bacteriano , Transcriptoma , RNA Polimerases Dirigidas por DNA/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , RNA Bacteriano/análise , RNA Bacteriano/biossíntese , RNA Bacteriano/genética , Transcriptoma/genética , Tuberculose/microbiologia , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Sítio de Iniciação de Transcrição , Fator sigma/metabolismo , Ribossomos/metabolismo , Biossíntese de ProteínasRESUMO
Transcriptional pauses mediate regulation of RNA biogenesis. DNA-encoded pause signals trigger pausing by stabilizing RNA polymerase (RNAP) swiveling and inhibiting DNA translocation. The N-terminal domain (NGN) of the only universal transcription factor, NusG/Spt5, modulates pausing through contacts to RNAP and DNA. Pro-pausing NusGs enhance pauses, whereas anti-pausing NusGs suppress pauses. Little is known about pausing and NusG in the human pathogen Mycobacterium tuberculosis (Mtb). We report that MtbNusG is pro-pausing. MtbNusG captures paused, swiveled RNAP by contacts to the RNAP protrusion and nontemplate-DNA wedged between the NGN and RNAP gate loop. In contrast, anti-pausing Escherichia coli (Eco) NGN contacts the MtbRNAP gate loop, inhibiting swiveling and pausing. Using CRISPR-mediated genetics, we show that pro-pausing NGN is required for mycobacterial fitness. Our results define an essential function of mycobacterial NusG and the structural basis of pro- versus anti-pausing NusG activity, with broad implications for the function of all NusG orthologs.
