In vitro characterization of 6S RNA release-defective mutants uncovers features of pRNA-dependent release from RNA polymerase in E. coli

6S RNA is a noncoding RNA that inhibits bacterial transcription by sequestering RNA polymerase holoenzyme ( Es70) in low-nutrient conditions. This transcriptional block can be relieved by the synthesis of a short product RNA ( pRNA) using the 6S RNA as a template. Here, we selected a range of 6S RNA release- defective mutants from a high diversity in vitro pool. Studying the release- defective variant R9- 33 uncovered complex interactions between three regions of the 6S RNA. As expected, mutating the transcriptional start site ( TSS) slowed and partially inhibited release. Surprisingly, additional mutations near the TSS were found that rescued this effect. Likewise, three mutations in the top strand of the large open bubble ( LOB) could considerably slow release but were rescued by the addition of upstream mutations found between a highly conserved "-35" motif and the LOB. Combining the three top strand LOB mutations with mutations near the TSS, however, was particularly effective at preventing release, and this effect could be further enhanced by inclusion of the upstream mutations. Overexpressing R9- 33 and a series of milder release- defective mutants in Escherichia coli resulted in a delayed entry into exponential phase together with a decrease in cell survival that correlated well with the severity of the in vitro phenotypes. The complex crosstalk observed between distinct regions of the 6S RNA supports a scrunching type model of 6S RNA release, where at least three regions of the 6S RNA must interact with Es70 in a cooperative manner so as to ensure effective pRNAdependent release.