Translocation and transcriptional arrest during transcript elongation by RNA polymerase II.
RNA polymerase II may stop transcription, or arrest, while transcribing certain DNA sequences. The molecular basis for arrest is not well understood, but a connection has been suggested between arrest and a transient failure of the polymerase to translocate along the template. We have investigated this question by monitoring the movement of RNA polymerase II along a number of templates, using exonuclease III protection as our assay. We found that normal transcription is accompanied by essentially coordinate movement of the active site and both the leading and trailing edges of the polymerase. However, as polymerase approaches an arrest site, translocation of the body of the polymerase stops while transcription continues, leading to an arrested complex in which the 3' end of the transcript is located much closer than normal to the front edge of the polymerase. Surprisingly, mutated arrest sites that no longer block transcription continue to direct the transient failure of polymerase translocation. As transcription proceeds through these sequences, the initially stationary polymerase moves forward 10-15 bases along the template in response to the addition of only 3 bases to the nascent RNA. Mutagenesis studies indicate that the sequences responsible for the transient block to polymerase movement are located downstream of the T-rich motif required for arrest. Our results indicate that blocking translocation is not sufficient to cause arrest.
Samkurashvili
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Department of Molecular Biology, Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Luse
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D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000235:genetics*; D018983:DNA Footprinting; D005090:Exodeoxyribonucleases / Q000378:metabolism; D008969:Molecular Sequence Data; D009038:Motion; D011485:Protein Binding; D012319:RNA Polymerase II / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis*; D014158:Transcription, Genetic
D012333:RNA, Messenger; D004247:DNA; D012319:RNA Polymerase II; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III