In vivo binding and hierarchy of assembly of the yeast RNA polymerase I transcription factors.

Transcription by RNA polymerase I in Saccharomyces cerevisiae requires a series of transcription factors that have been genetically and biochemically identified. In particular, the core factor (CF) and the upstream activation factor (UAF) have been shown in vitro to bind the core element and the upstream promoter element, respectively. We have analyzed in vivo the DNAse I footprinting of the 35S promoter in wild-type and mutant strains lacking one specific transcription factor at the time. In this way we were able to unambiguously attribute the protections by the CF and the UAF to their respective putative binding sites. In addition, we have found that in vivo a binding hierarchy exists, the UAF being necessary for CF binding. Because the CF footprinting is lost in mutants lacking a functional RNA polymerase I, we also conclude that the final step of preinitiation-complex assembly affects binding of the CF, stabilizing its contact with DNA. Thus, in vivo, the CF is recruited to the core element by the UAF and stabilized on DNA by the presence of a functional RNA polymerase I.

DNA protein-interactions at the Saccharomyces cerevisiae 35 S rRNA promoter and in its surrounding region.

This study represents a detailed analysis of the structural context of the RNA polymerase I promoter of Saccharomyces cerevisiae. We determined the presence of regularly spaced nucleosomes in the non-transcribed spacer (NTS) and found that five of them have well defined positions. We show that this nucleosome positioning is restricted to the region between the 35 S and 5 S rRNA promoters, beyond which a more delocalized chromatin structure is evident. A more refined analysis detects the DNA-protein interactions on the RNA polymerase I promoter at nucleotide resolution and provides the first in vivo footprints, attributable to factors like REB1, CF, UAF and an additional protection that seems to be sensitive to the topological context. Moreover, when this analysis is extended to different growth media (YPD versus YNB), some of these protections show a growth condition dependent behaviour.

Effect of polyols and sugars on heat-induced flavin dissociation in glucose oxidase.

A thermodynamic investigation was carried out on heat-induced flavin dissociation in Aspergillus niger glucose oxidase. Experimental measurements performed by difference spectroscopy showed that the dissociation of the FAD cofactors is a highly cooperative process and is probably related to the extended conformational changes resulting from protein unfolding. Microenvironmental modifications attained by the addition of polyhydric compounds (glycerol, fructose, sucrose and sorbitol) from 10 to 30% by weight were found to hinder the dissociation. The stabilizing effect provided by these substances was interpreted as a consequence of preferential exclusion phenomena, which are likely to be determined by the perturbation of the surface tension of water, in the case of sugars, or by the solvophobic effect, in the case of glycerol.