Short Transcripts of the Ternary Complex Provide Insight into RNA Polymerase II Elongational Pausing

• Published in: Journal of molecular biology 1995-10, Vol.252 (5), p.522-535
• Main Authors: Rasmussen, Eric B., Lis, John T.
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Cell Nucleus - genetics; Cells, Cultured; Cross-Linking Reagents - metabolism; DNA Primers; DNA-directed RNA polymerase; Drosophila melanogaster; Drosophila melanogaster - genetics; gapdh-1 gene; gene expression; Gene Expression Regulation; gene regulation; Genes, Insect; heat shock induction; heat shock proteins; Heat-Shock Response; hsp26 gene; hsp27 gene; hsp70 gene; HSP70 Heat-Shock Proteins - genetics; messenger RNA; Models, Genetic; Molecular Sequence Data; Polymerase Chain Reaction; RNA Polymerase II - chemistry; RNA Polymerase II - genetics; RNA Polymerase II - metabolism; RNA, Messenger - analysis; RNA, Messenger - metabolism; structural genes; transcription (genetics); Transcription, Genetic - genetics; transcriptional arrest; transcriptional pausing
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1995.0517

Expression of thehsp70 gene ofDrosophila melanogasteris 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 genein vivospanning 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 andhsp27) and two metabolic genes (Gapdh-1andGapdh-2), where paused polymerase were thought to be present, plus two other genes (Mtn andyp1), where polymerases 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.