Promoter clearance by T7 RNA polymerase. Initial bubble collapse and transcript dissociation monitored by base analog fluorescence.
Footprinting, fluorescence, and x-ray structural information from the initial, promoter-bound complex of T7 RNA polymerase 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, nontranscribed 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.
Liu
Cuihua
C
Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA.
Martin
Craig T
CT
D015075:2-Aminopurine / Q000737:chemistry; D020137:Base Pair Mismatch; D004247:DNA / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D005456:Fluorescent Dyes / Q000494:pharmacology*; D008954:Models, Biological; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014158:Transcription, Genetic; D014764:Viral Proteins
D005456:Fluorescent Dyes; D012333:RNA, Messenger; D014764:Viral Proteins; D015075:2-Aminopurine; D012313:RNA; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases