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 p ossesses an RNA polymerase II complex, elongationally engaged, but pau sed early in the transcription unit. In this study, we have used a pow erful new selection-amplification technique to analyze the RNA transcr ipts 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 in terrupted 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 pol ymerase pausing and release are discussed in light of these data. The increased sensitivity of our new technique also allowed us to investig ate ternary complexes associated with genes with much lower levels of engaged RNA polymerase. These included two of the small heat shock gen es (hsp26 and hsp27) and two metabolic genes (Gapdh-1 and Gapdh-2), wh ere paused polymerases were thought to be present, plus two other gene s (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 pre viously unknown sites of transcriptional termination, residing immedia tely upstream of the regions of polymerase pausing. (C) 1995 Academic Press Limited