In vivo assembly and single-molecule characterization of the transcription machinery from Shewanella oneidensis MR-1

Harnessing the new bioremediation and biotechnology applications offered by the dissimilatory metal-reducing bacteria, Shewanella oneidensis MR-1, requires a clear understanding of its transcription machinery, a pivotal component in maintaining vitality and in responding to various conditions, including starvation and environmental stress. Here, we have reconstituted the S. oneidensis RNA polymerase (RNAP) core in vivo by generating a co-overexpression construct that produces a long polycistronic mRNA encoding all of the core subunits (α, β, β′, and ω) and verified that this reconstituted core is capable of forming fully functional holoenzymes with the S. oneidensis σ factors σ⁷⁰, σ³⁸, σ³², and σ²⁴. Further, to demonstrate the applications for this reconstituted core, we report the application of single-molecule fluorescence resonance energy transfer (smFRET) assays to monitor the mechanisms of transcription by the S. oneidensis σ⁷⁰-RNAP holoenyzme. These results show that the reconstituted transcription machinery from S. oneidensis, like its Escherichia coli counterpart, "scrunches" the DNA into its active center during initial transcription, and that as the holoenzyme transitions into elongation, the release of σ⁷⁰ is non-obligatory.