Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
2021510 | Protein Expression and Purification | 2009 | 11 Pages |
Abstract
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, βâ², and Ï) and verified that this reconstituted core is capable of forming fully functional holoenzymes with the S. oneidensis Ï factors Ï70, Ï38, Ï32, and Ï24. 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 Ï70-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 Ï70 is non-obligatory.
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Authors
Natalie R. Gassman, Sam On Ho, You Korlann, Janet Chiang, Yim Wu, L. Jeanne Perry, Younggyu Kim, Shimon Weiss,