IN-VIVO TRANSCRIPTIONAL PAUSING AND CAP FORMATION ON 3 DROSOPHILA HEAT-SHOCK GENES

The regulation of many eukaryotic genes occurs at the level of transcr iptional elongation. On the uninduced hsp70 gene of Drosophila melanog aster, for example, an RNA polymerase II complex has initiated transcr iption but has paused early in elongation. In this study, we examine p ausing on hsp70 and two of the small heat shock genes (hsp27 and hsp26 ) at high resolution, using a technique that utilizes paramagnetic par ticle-mediated selection of terminated run-on transcripts. This techni que provides precise information on the distribution of RNA polymerase within each transcription unit. It also details the progression of 5' cap formation on the elongating transcripts. For each gene, we find p olymerases paused over a relatively narrow promoter-proximal region. T he regions are generally around 20 nucleotides wide, with two preferre d pausing positions spaced roughly 10 nucleotides apart or about one t urn of the helix. The bulk of capping occurs as transcripts pass betwe en 20 and 30 nucleotides in length. Interestingly, in the three genes examined here, elongational pausing and 5' cap formation appear largel y coincident.