LOCUS-SPECIFIC VARIATION IN PHOSPHORYLATION STATE OF RNA POLYMERASE-II IN-VIVO - CORRELATIONS WITH GENE ACTIVITY AND TRANSCRIPT PROCESSING

To investigate functional differences between RNA polymerases IIA and II0 (Pol IIA and Pol II0), with hypo- and hyperphosphorylated carboxy- terminal repeat domains (CTDs), respectively, we have visualized the i n vivo distributions of the differentially phosphorylated forms of Pol II on Drosophila polytene chromosomes. Using phosphorylation state-se nsitive antibodies and immunofluorescence microscopy with digital imag ing, we find Pol IIA and Pol II0 arrayed in markedly different, locus- and condition-specific patterns. Major ecdysone-induced puffs, for ex ample, stain exclusively for Pol II0, indicating that hyperphosphoryla ted Pol II is the transcriptionally active form of the enzyme on these genes. In striking contrast, induced heat shock puffs stain strongly for both Pol IIA and Pol II0, suggesting that heat shock genes are tra nscribed by a mixture of hypo- and hyperphosphorylated forms of Pol II . At the insertion sites of a transposon carrying a hybrid hsp70-lacZ transgene, we observe only Pol IIA before heat shock induction, consis tent with the idea that Pol II arrested on the hsp70 gene is form IIA. After a 90-sec heat shock, we detect heat shock factor (HSF) at the t ransposon insertion sites; and after a 5-min shock its spatial distrib ution on the induced transgene puffs is clearly resolved from that of Pol II. Finally, using antibodies to hnRNP proteins and splicing compo nents, we have discerned an apparent overall correlation between the p resence and processing of nascent transcripts and the presence of Pol II0.