Deciphering the Coupling State‐Dependent Transcription Termination in the <i>Escherichia coli</i> Galactose Operon

The distance between the ribosome and the RNA polymerase active centers, known as the mRNA loop length, is crucial for transcription-translation coupling. Despite the existence of multiple expressomes with varying mRNA loop lengths, their in vivo roles remain largely unexplored. This study examines the mechanisms governing transcription termination in the Escherichia coli galactose operon, revealing a crucial role in the transcription and translation coupling state. The operon utilizes both Rho-independent and Rho-dependent terminators. Our findings demonstrate that long-loop coupled transcription-translation complexes preferentially terminate at the upstream Rho-independent terminator, while short-loop complexes bypass it, terminating at the downstream Rho-dependent terminator. The efficiency of the Rho-independent terminator is enhanced by an extended U-track, suggesting a novel mechanism to overcome ribosome inhibition. These results uncover a new regulatory layer in transcription termination, challenging the traditional view of this process as random and highlighting a predetermined mechanism based on the coupling state. We propose that tandem terminators may function as regulatory checkpoints under fluctuating ribosome-RNAP coupling conditions, which can occur due to specific cellular states or factors affecting ribosome or RNAP binding efficiency. This suggests a previously overlooked mechanism that could refine transcription termination choices and expand our understanding of transcription regulation.