Analysis of core promoter sequences located downstream from the TATA element in the hsp70 promoter from Drosophila melanogaster.

TFIID recognizes multiple sequence elements in the hsp70 promoter of Drosophila. Here, we investigate the function of sequences downstream from the TATA element. A mutation in the initiator was identified that caused an eightfold reduction in binding of TFIID and a fourfold reduction in transcription in vitro. Another mutation in the +24 to +29 region was somewhat less inhibitory, but a mutation in the +14 to +19 region had essentially no effect. The normal promoter and the mutants in the initiator and the +24 to +29 region were transformed into flies by P element-mediated transformation. The initiator mutation reduced expression an average of twofold in adult flies, whereas the mutation in the +24 to +29 region had essentially no effect. In contrast, a promoter combining the two mutations was expressed an average of sixfold less than the wild type. The results suggest that the initiator and the +24 to +29 region could serve overlapping functions in vivo. Protein-DNA cross-linking was used to identify which subunits of TFIID contact the +24 to +29 region and the initiator. No specific subunits were found to cross-link to the +24 to +29 region. In contrast, the initiator cross-linked exclusively to dTAF230. Remarkably, dTAF230 cross-links approximately 10 times more efficiently to the nontranscribed strand than to the transcribed strand at the initiator.

Promoter-proximal pausing on the hsp70 promoter in Drosophila melanogaster depends on the upstream regulator.

RNA polymerase II pauses in the promoter-proximal region of many genes during transcription. In the case of the hsp70 promoter from Drosophila melanogaster, this pause is long-lived and occurs even when the gene is not induced. Paused polymerase escapes during heat shock when the transcriptional activator heat shock factor associates with the promoter. However, pausing is still evident, especially when induction is at an intermediate level. Yeast Gal4 protein (Gal4p) will induce transcription of the hsp70 promoter in Drosophila when binding sites for Gal4p are positioned upstream from the hsp70 TATA element. To further our understanding of promoter-proximal pausing, we have analyzed the effect of Gal4p on promoter-proximal pausing in salivary glands of Drosophila larvae. Using permanganate genomic footprinting, we observed that various levels of Gal4p induction resulted in an even distribution of RNA polymerase throughout the first 76 nucleotides of the transcribed region. In contrast, promoter-proximal pausing still occurs on endogenous and transgenic hsp70 promoters in salivary glands when these promoters are induced by heat shock. We also determined that mutations introduced into the region where the polymerase pauses do not inhibit pausing in a cell-free system. Taken together, these results indicate that promoter-proximal pausing is dictated by the regulatory proteins interacting upstream from the core promoter region.

Modification of the thermoluminescence properties of Ca(2+) depleted Photosystem II membranes by the 23 kDa extrinsic polypeptide and by oligocarboxylic acids.

Photosystem II membranes were isolated from chloroplasts of pokeweed (Phytolacca americana) and rendered deficient in Ca(2+), an inorganic cofactor of photosynthetic water oxidation. The thermoluminescence properties of such membranes were found to depend on the Ca(2+)-depleting method used. This feature was analyzed with respect to the thermoluminescence emission that accompanied the recombination reaction between the reduced acceptor QA (-) and the oxidant of the S2 state. It was determined that the differences observed among various preparations of Ca(2+)-depleted membranes were attributable to the presence or absence of the extrinsic 23 kDa polypeptide on the membranes. The binding of this polypeptide to Ca(2+)-depleted membranes devoid of the 17 and 23 kDa extrinsic polypeptides caused the thermoluminescence to be emitted at a higher temperature due to a further stabilization of an already abnormally stable S2 state. Addition of the chelators EDTA or EGTA and of citrate brought about a similar response. The conditions required for the upshift of the emission temperature of thermoluminescence strongly resembled those identified by Boussac et al. (FEBS Lett. 277 (1990) 69-74) as responsible for modifying the EPR multiline signal from the S2 state of Ca(2+)-depleted PS II membranes. Consistent with the authors' interpretation of the reason for this modification, we conclude that the elevated emission temperature of the thermoluminescence emission reflects an abnormal ligand environment of the Mn-center in PS II that may be created by a direct ligation of the added agents to Mn. Evidence is also presented that the return to a normal S2 after an addition of Ca(2+) occurs via yet another condition of S2 which, in terms of its thermoluminescence properties, resembles that of Ca(2+)-depleted membranes before addition of modifying agents, but is not identical to it.