Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 277, Issue 4, Pages 2725-2731Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M108856200
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- NIGMS NIH HHS [1R01GM55002] Funding Source: Medline
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Footprinting, fluorescence, and x-ray structural information from the initial, promoter-bound complex of T7 RNA polymerasc describes the very beginning of the initiation of transcription, whereas recent fluorescence and biochemical studies paint a preliminary picture of an elongation complex. The current work focuses on the transition from an initially transcribing, promoter-bound complex to an elongation complex clear of the promoter. Fluorescence quenching is used to follow the melted state of the DNA bubble, and a novel approach using a locally mismatched fluorescent base analog reports on the local structure of the heteroduplex. Fluorescent base analogs placed at positions -2 and -1 of the promoter indicate that this initially melted, non-transcribed region remains melted as the polymerase translocates through to position +8. In progressing to position +9, this region of the DNA bubble begins to collapse. Probes placed at positions +1 and +2 of the template strand indicate that the 5' end of the RNA remains in a heteroduplex as the complex translocates to position +10. Subsequent translocation leads to sequential dissociation of the first 2 bases of the RNA. These results show that the initially transcribing complex bubble can reach a size of up to 13 base pairs and a maximal heteroduplex length of 10 base pairs. They further indicate that initial bubble collapse precedes dissociation of the 5' end of the RNA.
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