In vivo regulation of bacterial Rho-dependent transcription termination by the nascent RNA.

Bacterial Rho is a RNA-dependent ATPase that functions in the termination of transcription. The in vivo nature of the bacterial Rho-dependent terminators, as well as the mechanism of the Rho-dependent termination process, are not fully understood. Here, we measured the in vivo termination efficiencies of 72 Rho-dependent terminators in Escherichia coli by systematically performing qRT-PCR analyses of cDNA prepared from mid-log phase bacterial cultures. We found that these terminators exhibited a wide range of efficiencies, and many behaved differently in vivo compared to the predicted or experimentally determined efficiencies in vitro. Rho-utilization sites (rut sites) present in the RNA terminator sequences are characterized by the presence of C-rich/G-poor sequences or C > G bubbles. We found that weaker terminators exhibited a robust correlation with the properties (size, length, density, etc.) of these C > G bubbles of their respective rut sites, while stronger terminators lack this correlation, suggesting a limited role of rut sequences in controlling in vivo termination efficiencies. We also found that in vivo termination efficiencies are dependent on the rates of ATP hydrolysis as well as Rho-translocation on the nascent RNA. We demonstrate that weaker terminators, in addition to having rut sites with diminished C > G bubble sizes, are dependent on the Rho-auxiliary factor, NusG, in vivo. From these results, we concluded that in vivo Rho-dependent termination follows a nascent RNA-dependent pathway, where Rho-translocation along the RNA is essential and rut sequences may recruit Rho in vivo, but Rho-rut binding strengths do not regulate termination efficiencies.

Rho-dependent transcription termination regulates the toxin-antitoxin modules of cryptic prophages to silence their expression in Escherichia coli.

Bacterial Rho-dependent transcription termination regulates many physiological processes. Here, we report that it controls the expression of toxin-antitoxin (TA) modules of cryptic prophages in E. coli. Microarray profiles of Rho mutants showed upregulation of genes of the CP4-6 and CP4-44 prophages, including their TA modules, that were validated by RT-qPCR. Analysis of the in vivo termination efficiency and the mRNA sequences of these prophages revealed the presence of many Rho-dependent terminators. The prophage TA modules exhibited synthetic lethality with the Rho mutants, indicating functional involvement of Rho-dependent termination in controlling these modules. Rho-dependent termination does not regulate most of the chromosomal TA modules. We conclude that Rho-dependent termination specifically silences the TA modules of prophages, thereby augmenting bacterial innate immunity.