RNA polymerase stalling-derived genome instability underlies ribosomal antibiotic efficacy and resistance evolution

In bacteria, the precise coordination of DNA replication, transcription and translation is mediated by dynamic interactions among the corresponding macromolecular machineries, playing a pivotal role in maintaining cellular homeostasis. Here we showed that such coordination could be hijacked by ribosome antibiotics to trigger secondary damage predominantly contributing to their efficacy, via a yet overlooked reverse-central-dogma pathway. Through the utilization of a self-establishing transcription dynamics profiling method, complemented by genetic and biochemical approaches, we unveil that the disruption of transcription-translation coupling leads to premature stalling of RNA polymerase (RNAP) at genome scale, subsequently triggering extensive genomic instability. Moreover, a distinct subpopulation exhibits hyperactivation of the SOS response, facilitating an inducible evolutionary path towards genetic resistance characterized by a unique mutation spectrum. Our findings reveal the emergence of secondary drug damage resulting from network disorder, and establish a framework to understand antibiotic efficacy and induced mutagenesis from a systems biology perspective. To investigate the secondary effect of gentamicin treatment, which is transcription elongation chaos and mutagensis, we performed RNA-seq and 3' -end-seq to profile transcription dynamics and genome re-sequencing to identify mutations.