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The uninduced Drosophila hsp70 gene is poised for rapid activation. Here we examine the rapid changes upon heat shock in levels and location of heat shock factor (HSF), RNA polymerase II (Pol II) and its phosphorylated forms, and the Pol II kinase P-TEFb on hsp70 in vivo by using both real-time PCR assays of chromatin immunoprecipitates and polytene chromosome immunofluorescence. These studies capture Pol II recruitment and progression along hsp70 and reveal distinct spatial and temporal patterns of serine 2 and serine 5 phosphorylation: in uninduced cells, the promoter-paused Pol II shows Ser5 but not Ser2 phosphorylation, and in induced cells the relative level of Ser2-P Pol II is lower at the promoter than at regions downstream. An early time point of heat shock activation captures unphosphorylated Pol II recruited to the promoter prior to P-TEFb, and during the first wave of transcription Pol II and the P-TEFb kinase can be seen tracking together across hsp70 with indistinguishable kinetics. Pol II distributions on several other genes with paused Pol II show a pattern of Ser5 and Ser2 phosphorylation similar to that of hsp70. These studies of factor choreography set important limits in modeling transcription regulatory mechanisms.Recent studies of posttranslational modifications of, and factor interactions with, the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) have implicated this region of Pol II in productive transcriptional elongation and the coupling of transcription and pre-mRNA processing (for reviews see references 32 and 37). The CTD is highly conserved among eukaryotes, containing multiple repeats of a consensus heptad YSPTSPS; mammalian Pol II CTD consists of 52 repeats, yeast consists of 25 to 26 repeats, while Drosophila has 42 repeats (1, 6). This large flexible arm of Pol II appears to serve as a docking site for, or to stimulate the recruitment of, an orchestrated assembly of factors involved in pre-mRNA capping, splicing, and 3Ј polyadenylation at different stages in production of the nascent transcript (5,9,13,14,20,26). One way this coordination has been proposed to occur is through the known phosphorylation of serines 2 and 5 (Ser2-P and Ser5-P, respectively) of the heptad repeat (52, 56). Cdk7, the kinase subunit of general transcription factor TFIIH, has been shown to phosphorylate the CTD at Ser5, an event proposed to occur early in the transcription cycle (12,24,57). This in turn appears to influence the association and activity of the capping machinery (5,15,18,27,28,42,45). Positive transcription elongation factor b (P-TEFb) is able to phosphorylate the CTD at Ser2 and, under certain conditions, Ser5 (25, 38, 57). P-TEFb is a kinase composed of the proteins cyclin T (CycT) and cdk9 and is known to be recruited upon gene activation, overcoming the negative effects of factors like Spt5 and negative elongation factor (50) and aiding in the transition from transcription initiation to elongation (36). Cdk8, a component of the coactivator complex Mediator, h...

RNA polymerase II (Pol II) transcription through nucleosomes is facilitated in vitro by the protein complex FACT (Facilitates Chromatin Transcription). Here we show that FACT is associated with actively transcribed Pol II genes on Drosophila polytene chromosomes. FACT displays kinetics of recruitment and of chromosome tracking in vivo similar to Pol II and elongation factors Spt5 and Spt6. Interestingly, FACT does not colocalize with Pol III-transcribed genes, which are known to undergo nucleosome transfer rather than disassembly in vitro. Our observations are consistent with FACT being restricted to transcription that involves nucleosome disassembly mechanisms.