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From an initially transcribing to a processively elongating T7 RNA polymerase: Fluorescence resonance energy transfer (FRET) tests of structural models
Although structurally more simple, T7 RNA polymerase shares functional similarities with the more complex, multi-subunit RNA polymerases. Upon recognition and binding to its short promoter DNA, T7 RNA polymerase induces DNA bending that is centered close to the transcription start site as evidenced by gel-shift assays and by recent FRET distance measurements. Crystal structures of promoter-bound and initially-transcribing complexes of T7 RNA polymerase do not provide any information on the path of the downstream DNA, a region that is seen only in the elongation complex crystal structures. A complete model for the initiation complex can be created by alignment of the C-terminal regions of the protein structures from both initiation and elongation complexes and then transferring the downstream DNA from the elongation complex onto the initiation complex. Using 5-carboxytetramethylrhodamine (TAMRA) placed at the upstream -17 position on the nontemplate strand and fluorescein-5-isothiocyanate (FITC) at various positions downstream on the template strand, we have confirmed this model by fluorescence resonance energy transfer (FRET) distance measurements with or without adding 3'dGTP. Data confirm both model orientation and helical phasing of the downstream DNA. T7 RNA polymerase undergoes a dramatic structural rearrangement in transitioning from initiation to elongation. Two models have been proposed for the structure of promoter-bound intermediates late in the transition: (1) structural rearrangement of the protein is nearly complete requiring a substantial rotation of the N-terminal platform (left-handed 140° rotation); (2) N-terminal rigid body subdomain primarily translates with only a slight rotation - the large right-handed rotation of 220° occurs only after promoter release. FRET distance measurements for complexes stalled at position +8 provide strong support for the latter model. Finally, FRET distances for complexes chased to position +9 and to position +10 were obtained to provide preliminary information on the spatial location of the released upstream promoter DNA in elongation complex, a region that is not resolved in the elongation complex crystal structure. In addition, complexes were also translocated to position +11 and then challenged with a tight-binding promoter sink to ensure that release happens and investigate whether the released promoter returns to an extended linear duplex DNA conformation.
Turingan, Rosemary Sanchez, "From an initially transcribing to a processively elongating T7 RNA polymerase: Fluorescence resonance energy transfer (FRET) tests of structural models" (2007). Doctoral Dissertations Available from Proquest. AAI3254946.