Short transcripts of the ternary complex provide insight into RNA polymerase II elongational pausing.
Expression of the hsp70 gene of Drosophila melanogaster is controlled at the level of transcript elongation. In the uninduced state, hsp70 possesses an RNA polymerase II complex, elongationally engaged, but paused early in the transcription unit. In this study, we have used a powerful new selection-amplification technique to analyze the RNA transcripts associated with such "paused polymerases" under non-heat shock and heat shock-induced conditions. They reveal a region of pausing on the uninduced gene in vivo spanning from +21 to +35. This region is interrupted by an area of low polymerase density, centered at about +26. Upon induction, an accumulation of short transcripts, similar in size to those associated with the paused complex, was seen. Models for polymerase pausing and release are discussed in light of these data. The increased sensitivity of our new technique also allowed us to investigate ternary complexes associated with genes with much lower levels of engaged RNA polymerase. These included two of the small heat shock genes (hsp26 and hsp27) and two metabolic genes (Gapdh-1 and Gapdh-2), where paused polymerases were thought to be present, plus two other genes (Mtn and yp1), where polymerase pausing has not been detected in the past. In both of the small heat shock genes, we found evidence of previously unknown sites of transcriptional termination, residing immediately upstream of the regions of polymerase pausing.
Rasmussen
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Section of Biochemistry Molecular and Cell Biology, Cornell University, Ithaca New York, 14853, USA.
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D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000235:genetics; D002478:Cells, Cultured; D003432:Cross-Linking Reagents / Q000378:metabolism; D017931:DNA Primers; D004331:Drosophila melanogaster / Q000235:genetics*; D005786:Gene Expression Regulation; D017344:Genes, Insect; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D018869:Heat-Shock Response; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012319:RNA Polymerase II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics*
D003432:Cross-Linking Reagents; D017931:DNA Primers; D018840:HSP70 Heat-Shock Proteins; D012333:RNA, Messenger; D012319:RNA Polymerase II