The regulation of many eukaryotic genes occurs at the level of transcriptional elongation. On the uninduced hsp7O gene of Drosophila melanogaster, for example, an RNA polymerase I complex has initiated transcription but has paused early in elongation. In this study, we examine pausing on hsp7O and two of the small heat shock genes (hsp27 and hsp26) at high resolution, using a technique that utilizes paramagnetic particle-mediated selection of terminated run-on transcripts. This technique provides precise information on the distribution ofRNA polymerase within each transcription unit. It also details the progression of 5' cap formation on the elongating transcripts. For each gene, we find polymerases paused over a relatively narrow promoter-proximal region. The regions are generally around 20 nucleotides wide, with two preferred pausing positions spaced roughly 10 nucleotides apart or about one turn of the helix. The bulk ofcapping occurs as transcripts pass between 20 and 30 nucleotides in length. Interestingly, in the three genes examined here, elongational pausing and 5' cap formation appear largely coincident.The transcription of eukaryotic genes is a complex, multistep process. Often, its regulation is assumed to lie mainly in the initiation event, but regulation could potentially be accomplished at any of the numerous steps leading to a full-length transcript: from the binding of RNA polymerase and its accessory factors, through initiation and elongation, to proper termination. For many genes, the control oftranscription occurs after initiation. A blockage or pause during elongation prevents formation of the full-length message; relief of that blockage is required for high-level expression of the gene. This type of transcriptional control has been found in a variety of eukaryotic genes and organisms, including human immunodeficiency virus type 1(1), human and murine c-myc (2-4) and ADA (5, 6), and Drosophila hsp7O (7). (See ref. 8 for a review.)Genes that utilize elongational blockage for transcriptional control have as a result a greater density of RNA polymerase on their 5' ends than on latter regions. Several genes in Drosophila melanogaster have been identified that show such a polymerase distribution. These include heat-inducible (hsp7O, hsp26), metabolic (Gapdh-1, Gapdh-2), and structural genes (f31-Tubulin) (9); thus, the phenomenon appears relatively general. The most thoroughly studied of these is hsp7O. In the uninduced state, it has roughly one RNA polymerase II complex present in the promoter-proximal region of each gene (10,11). This polymerase has initiated transcription but has paused early in the elongational process (7). Upon induction by heat shock, the rate of escape of these paused polymerases into elongation rises dramatically, leading to a >100-fold increase in hsp7O transcription (12). Interestingly, elongational pausing remains the rate-limitingThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "adv...